Crystalline forms of a cd73 inhibitor and uses thereof

ABSTRACT

Crystalline forms of the compound of Formula (I), which modulates the conversion of AMP to adenosine by 5′-nucleotidase, ecto, and compositions containing the compound and methods for preparing the crystalline forms, are described herein. The use of such crystalline form of Formula (I) and compositions for the treatment and/or prevention of a diverse array of diseases, disorders and conditions, including cancer and immune-related disorders, that are mediated by 5′-nucleotidase, ecto is also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 63/040,277, filed Jun. 17, 2020, which isincorporated by reference herein in its entirety for all purposes.

FIELD

Provided herein are, for example, crystalline forms of a compound andcompositions for inhibition of adenosine by 5′-nucleotidase, ecto, alsoknown as CD73, and pharmaceutical compositions comprising same. Alsoprovided herein are, for example, methods of treating or preventing adisease, disorder or condition, or a symptom thereof, mediated byinhibition of adenosine by 5′-nucleotidase, ecto.

BACKGROUND OF THE DISCLOSURE

Ectonucleotides catalyze the conversion of ATP to adenosine, anendogenous modulator that impacts multiple systems, including the immunesystem, the cardiovascular system, the central nervous system, and therespiratory system. Adenosine also promotes fibrosis in a variety oftissues. In the first step of the production of adenosine,ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also known asCD39 (Cluster of Differentiation 39), hydrolyzes ATP to ADP, and thenADP to AMP. In the next step, AMP is converted to adenosine by5′-nucleotidase, ecto (NT5E or 5NT), also known as CD73 (Cluster ofDifferentiation 73).

The enzymatic activities of CD39 and CD73 play strategic roles incalibrating the duration, magnitude, and chemical nature of purinergicsignals delivered to various cells (e.g., immune cells). Alteration ofthese enzymatic activities can change the course or dictate the outcomeof several pathophysiological events, including cancer, autoimmunediseases, infections, atherosclerosis, and ischemia-reperfusion injury,suggesting that these ecto-enzymes represent novel therapeutic targetsfor managing a variety of disorders.

CD73 inhibition with monoclonal antibodies, siRNA, or small moleculesdelays tumor growth and metastasis (Stagg, J. (2010) PNAS U.S.A.107:1547-52). For example, anti-CD73 antibody therapy was shown toinhibit breast tumor growth and metastasis in animal models (Stagg, J.(26 Jan. 2010) PNAS U.S.A, 107(4):1547-52). In addition, the use ofantibodies that specifically bind CD73 has been evaluated for thetreatment of bleeding disorders (e.g., hemophilia) (U.S. Pat. No.9,090,697). There have been several efforts to develop therapeuticallyuseful CD73 small molecule inhibitors. However, the development of smallmolecules has been hampered due to, for example, less than idealphysical and metabolic stability.

The compound(((((2R,3S,4R,5R)-5-(6-chloro-4-(((S)-1-(2-fluorophenyl)ethyl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)-phosphoryl)methyl)phosphonicacid, designated herein as Compound I, is a potent and selectivesmall-molecule inhibitor of CD73. In view of the role played by CD73 incancer, as well as a diverse array of other diseases, disorders andconditions, and the current lack of CD73 inhibitors available to medicalpractitioners, there is a need for stable crystalline forms of CompoundI, as well as compositions and methods associated therewith.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure relates to crystalline forms of a compound thatmodulates the conversion of AMP to adenosine by 5′-nucleotidase, ecto(NT5E or 5NT; also known as CD73), and compositions (e.g.,pharmaceutical compositions) comprising the compound. Such a compound(in a crystalline form), including methods of preparation, methods ofuse, and compositions are described in detail below.

In one aspect, the present disclosure provides a crystalline form of acompound having Formula (I):

wherein the crystalline form is any one of crystalline Forms I to VI,each of which is characterized by an X-ray powder diffraction (XRPD)pattern as described herein.

In another aspect, the present disclosure provides a process forpreparing a crystalline form of a compound of Formula (I), the processincluding:

-   -   a) forming a first mixture comprising a compound of Formula (I)        and a solvent at a temperature of at least 20° C.; and    -   b) adding an anti-solvent to the first mixture to form a second        mixture,    -   or    -   a) forming a first mixture comprising a compound of Formula (I)        and a solvent at a temperature of at least 20° C.;    -   c) cooling the first or second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline form of        Formula (I),    -   wherein the solvent is a C₁₋₄ alkyl alcohol, a di-(C₁₋₄ alkyl)        ether, a 5-6 membered cyclic ether, acetic acid, or water; and        the anti-solvent is a C₅₋₇alkane, C₁₋₄alkyl alcohol, a        di-(C₁₋₄alkyl) ether,        -   a 5-6 membered cyclic ether, a di-(C₁₋₄alkyl) ketone, C₁₋₄            alkyl-C(O)O—C₁₋₄alkyl, or an aromatic hydrocarbon solvent,            provided that the solvent and anti-solvent are not each a            C₁₋₄alkyl alcohol, a di-(C₁₋₄alkyl) ether, or a 5-6 membered            cyclic ether.

The present disclosure also relates to the use of the crystalline formsof such a compound and compositions for the treatment and/or preventionof a diverse array of diseases, disorders and conditions mediated, inwhole or in part, by CD73. CD73 inhibitors have been linked to thetreatment of a diverse array of disorders, including cancer, fibrosis,neurological and neurodegenerative disorders (e.g., depression andParkinson's disease), cerebral and cardiac ischemic diseases,immune-related disorders, and disorders with an inflammatory component.[See, e.g., Sorrentino et al (2013) Oncolmmunol, 2:e22448, doi:10.4161/onci.22448; and Regateiro et al. (2012) Clin. Exp. Immunol,171:1-7]. In particular embodiments, the crystalline forms of thecompound described herein can be formulated in a manner to inhibit theimmunosuppressive activity and/or the anti-inflammatory activity ofCD73, and are useful as therapeutic or prophylactic therapy when suchinhibition is desired. Unless otherwise indicated, when uses refer tothe compound (or to the crystalline form of the compound) describedherein, it is to be understood that such compound may be in a formappropriate for delivery (e.g., a pharmaceutical composition).

In some embodiments, the present disclosure contemplates methods fortreating or preventing cancer in a subject (e.g., a human) comprisingadministering to the subject a therapeutically effective amount of acrystalline form of a compound of Formula (I). The present disclosureincludes methods of treating or preventing a cancer in a subject byadministering to the subject a crystalline form of a compound of Formula(I) in an amount effective to reverse, stop or slow the progression ofCD73-mediated immunosuppression.

Examples of the cancers that can be treated using the crystalline formof a compound of Formula (I) and compositions described herein include,but are not limited to: cancers of the prostate, such as metastaticcastration resistant prostate cancer, colorectum, pancreas, cervix,stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck,skin (including melanoma and basal carcinoma), mesothelial lining, whiteblood cell (including lymphoma and leukemia) esophagus, breast, muscle,connective tissue, lung (including small-cell lung carcinoma andnon-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone;glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma,sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, andtesticular seminoma. In some embodiments of the present disclosure, thecancer is melanoma, colon cancer, pancreatic cancer, breast cancer,prostate cancer, lung cancer, leukemia, a brain tumor, lymphoma,sarcoma, ovarian cancer, or Kaposi's sarcoma. Cancers that arecandidates for treatment with a crystalline form of a compound ofFormula (I) and compositions of the present disclosure are discussedfurther hereafter.

The present disclosure contemplates methods of treating a subjectreceiving a bone marrow transplant or peripheral blood stem celltransplant by administering a therapeutically effective amount of acrystalline form of a compound of Formula (I) sufficient to increase thedelayed-type hypersensitivity reaction to tumor antigen, delay thetime-to-relapse of post-transplant malignancy, increase relapse-freesurvival time post-transplant, and/or increase long-term post-transplantsurvival.

In certain embodiments, the present disclosure contemplates methods fortreating or preventing an infective disorder (e.g., a viral infection)in a subject (e.g., a human) comprising administering to the subject atherapeutically effective amount of a crystalline form of a compound ofFormula (I). In some embodiments, the infective disorder is a viralinfection (e.g., a chronic viral infection), a bacterial infection, afungal infection, or a parasitic infection. In certain embodiments, theviral infection is human immunodeficiency virus or cytomegalovirus.

In still other embodiments, the present disclosure contemplates methodsfor treating and/or preventing immune-related diseases, disorders andconditions; diseases having an inflammatory component; as well asdisorders associated with the foregoing; with a crystalline form of acompound of Formula (I). Examples of immune-related diseases, disordersand conditions are described hereafter.

Other diseases, disorders and conditions that can be treated orprevented, in whole or in part, by modulation of CD73 activity arecandidate indications for a crystalline form of a compound of Formula(I) as described herein.

The present disclosure further contemplates the use of the crystallineform of a compound of Formula (I) described herein in combination withone or more additional agents. The one or more additional agents mayhave some CD73-modulating activity and/or they may function throughdistinct mechanisms of action. In some embodiments, such agents compriseradiation (e.g., localized radiation therapy or total body radiationtherapy) and/or other treatment modalities of a non-pharmacologicalnature. When combination therapy is utilized, the crystalline form of acompound of Formula (I) and the one additional agent(s) may be in theform of a single composition or multiple compositions, and the treatmentmodalities can be administered concurrently, sequentially, or throughsome other regimen. By way of example, the present disclosurecontemplates a treatment regimen wherein a radiation phase is followedby a chemotherapeutic phase. The combination therapy can have anadditive or synergistic effect. Other benefits of combination therapyare described hereafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an X-ray powder diffraction (XRPD) pattern of cystallineForm I of the compound of Formula (I).

FIG. 2 shows a differential scanning calorimetry (DSC) plot ofcystalline Form I of the compound of Formula (I).

FIG. 3 shows an X-ray powder diffraction (XRPD) pattern of cystallineForm II of the compound of Formula (I).

FIG. 4 shows a differential scanning calorimetry (DSC) plot ofcystalline Form II of the compound of Formula (I).

FIG. 5 shows an X-ray powder diffraction (XRPD) pattern of cystallineForm III of the compound of Formula (I).

FIG. 6 shows a differential scanning calorimetry (DSC) plot ofcystalline Form III of the compound of Formula (I).

FIG. 7 shows an X-ray powder diffraction (XRPD) pattern of cystallineForm IV of the compound of Formula (I).

FIG. 8 shows an X-ray powder diffraction (XRPD) pattern of crystallineForm V of the compound of Formula (I).

FIG. 9 shows a differential scanning calorimetry (DSC) plot ofcystalline Form V of the compound of Formula (I).

FIG. 10 shows an X-ray powder diffrations (XRPD) pattern of crystallineForm VI of the compound of Formula (I).

FIG. 11 shows a differential scanning calorimetry (DSC) plot ofcrystalline Form VI of the compound of Formula (I).

FIG. 12A-D shows the X-ray powder diffraction (XRPD) pattern of solidsrecovered from competitive slurry experiments using varying amounts ofethanol and ethyl acetate. FIG. 12A shows the XRPD pattern of Form Ireference, Form V starting material, Form II reference, and the materialrecovered from slurrying a mixture of Forms I, II and V in 100% EtOH attime 0, and after 1 day; and the material recovered from slurrying amixture of Forms I, II and V in 80% Ethanol: 20% EtOAc at time 0, andafter 1 day, and 2 days. FIG. 12B shows the XRPD pattern of Form Ireference, Form V starting material, Form II reference, and the materialrecovered from slurrying a mixture of Forms I, II and V in 67% EtOH: 33%EtOAc at time 0, and after 1 day and 2 days; and the material recoveredfrom slurrying a mixture of Forms I, II and V in 50% Ethanol: 50% EtOAcat time 0, and after 1 day. FIG. 12C shows the XRPD pattern of Form Ireference, Form V starting material, Form II reference, and the materialrecovered from slurrying a mixture of Forms I, II and V in 33% EtOH: 67%EtOAc at time 0, and after 1 day and 2 days. FIG. 12D shows the XRPDpattern of Form I reference, Form V starting material, Form IIreference, and the material recovered from slurrying a mixture of FormsI, II and V in 20% EtOH: 80% EtOAc at time 0, and after 2 days; and thematerial recovered from slurrying a mixture of Forms I, II and V in 100%EtOAc at time 0, and after 2 days.

FIG. 13 depicts the solubility profiles of Forms I (diamond), II(square) and V (triangle) in EtOH/EtOAc solvent systems at 20° C.

FIG. 14 depicts a polymorphic relationship map based on observations ofslurry experiments using EtOH:EtOAc solvent systems. The conditionsdepicted in the relationship map are merely exemplary, and are notintended to represent all routes that can be used to generate thedepicted crystalline forms.

FIG. 15 shows the solubility profile of Form I at 20° C. (square), FormI at 35° C. (circle), a mixture of Form II and V at 20° C. (diamond),and a mixture of Form II and V at 35° C. (triangle) in EtOH/heptanesolvent systems.

FIG. 16 shows a dynamic vapor sorption (DVS) isotherm of Form I of thecompound of Formula (I).

FIG. 17 shows a dynamic vapor sorption (DVS) isotherm of Form II of thecompound of Formula (I).

DETAILED DESCRIPTION OF THE DISCLOSURE

Before the present disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology such as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

As used herein, the term “a crystalline form of a compound of Formula(I)” refers to any of the crystalline forms of the noted compound asdescribed herein. The crystalline form can be, however, formulated to aliquid, gel, or ointment, for example, for ease of administration to asubject. In particular, with reference to a method involving theadministration of a crystalline form of a compound of Formula (I), themethod is meant to include administration of a liquid formulation thatis prepared using the crystalline form of a compound of Formula (I).

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Further,the dates of publication provided may be different from the actualpublication dates, which may need to be independently confirmed.

I. General

The compound(((((2R,3S,4R,5R)-5-(6-chloro-4-(((S)-1-(2-fluorophenyl)ethyl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonicacid, represented by Formula (I) is a potent inhibitor of CD73:

The present disclosure results from the surprising discoveries of thecrystalline forms of the compound of Formula (I), advantages attributedto the forms as described herein, and processes for making thecrystalline forms. Crystalline materials are generally more stablephysically and chemically. The superior stability of crystallinematerial may make them more suitable to be used in the final dosage formas shelf life of the product is directly correlated with stability.

The manufacturing process of a compound may also play a role inselecting a desired polymorphic form. As one example, compounds thatexhibit transient solubility due to the tendency of certain polymorphicforms to precipitate from solution can make maximizing the efficiency ofa manufacturing protocol a challenge. In such cases, an understanding ofthe solubility profiles of the polymorphic forms of a compound can beimportant for process development. For example, the solubility profilescan inform decisions regarding solvent systems that can be utilized tosolubilize all polymorphic forms of the desired compound such thatpurifying techniques, such as, for example, polishing filtration, can beconducted with minimal loss of the desired compound due to precipitationon the filter. Subsequent to such purifying techniques, the solubilityprofiles of the various polymorphic forms may inform decisions regardingtechniques that are useful to precipitate the desired compound fromsolution such that the overall yield is maximized (i.e., controlling thesolvent conditions to induce precipitation of the most stablepolymorphic form under those conditions). Certain polymorphic forms mayalso have physical properties that allow for them to be handled moreeasily during the manufacturing process. For example, crystallinemorphology (e.g., needles, plates or prisms) can influence the ease offiltration and drying protocols. Additional physical properties such as,for example, hygroscopicity, bulk density and flowability may providecertain benefits to the manufacturing process. Additionally, acrystallization step in active pharmaceutical ingredient (API)processing also provides an opportunity to enhance the drug substancepurity by removing impurities (e.g., such as those in the processingsolvent). Finally, certain polymorphic forms may be selected due tosuitability for pharmaceutical applications, e.g., having a residualsolvent content that is safe for patient administration (e.g., orally orparenterally).

II. Definitions

Unless otherwise indicated, the following terms are intended to have themeaning set forth below. Other terms are defined elsewhere throughoutthe specification.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated (i.e., C₁₋₄ means one tofour carbons). Alkyl can include any number of carbons, such as C₁₋₂,C₁₋₃, C₁₋₄, C₂₋₃, and C₃₋₄. For example, C₁₋₄ alkyl includes, but is notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl.

“Hydrate” refers to a complex formed by the combining of the compound ofFormula (I) and water. The term includes stoichiometric as well asnon-stoichiometric hydrates.

“Solvate” refers to a complex formed by the combining of the compound ofFormula (I) and a solvent. The term includes stoichiometric as well asnon-stoichiometric solvates. Exemplary solvents that form solvatesinclude, but are not limited to methanol, ethanol, isopropanol, DMSO,ethyl acetate, acetic acid, acetonitrile, and methyl tert-butyl ether.In some embodiments, the crystalline form of the compound of Formula (I)is an acetonitrile, ethanol, ethylacetate or methyl tert-butyl ethersolvate.

“Desolvated” refers to a form of the compound of Formula (I) that is asolvate as described herein, and from which solvent molecules have beenpartially or completely removed. Desolvation techniques to producedesolvated forms include, without limitation, exposure of a Form(solvate) of the compound of Formula (I) to a vacuum, subjecting thesolvate to elevated temperature, exposing the solvate to a stream ofgas, such as air or nitrogen, washing or slurrying the solvate in adifferent solvent that has less propensity to bind, or any combinationthereof. Thus, a desolvated form of the compound of Formula (I) can becompletely without solvent molecules, or partially solvated whereinsolvent molecules are present in stoichiometric or non-stoichiometricamounts.

“Alcohol” refers to a solvent having a hydroxy group. Representativealcohols can have any suitable number of carbon atoms, such as C₁-C₆,and any suitable number of hydroxy groups, such as 1-3. Exemplaryalcohols include, but are not limited to, methanol, ethanol, n-propanol,i-propanol, etc.

“Crude” refers to a mixture including a desired compound (e.g., thecompound of formula (I)) and at least one other species (e.g., asolvent, a reagent such as an acid or base, a starting material, or abyproduct of a reaction giving rise to the desired compound).

Suitable solvents described herein, refer to solvents characterized withhigh solubility of the compound of Formula (I) at a concentration of atleast about 50 mg/mL at 55-60° C. Anti-solvents, are generallyconsidered ‘poor solvents’, refer to solvents characterized with lowsolubility of the compound of Formula (I) at a concentration of lessthan about 50 mg/mL at 55-60° C. While anti-solvents may be poor fordissolving the compound, they can be well suited for crystallizationpurposes.

“Precipitating” refers to the process of causing a compound in asolution to coalesce into a solid form of the substance (i.e., aprecipitate). The entirety of a compound in a solution, or any fractionthereof, can be caused to precipitate. The solid form of the substancecan be amorphous or crystalline.

“Crystalline form” refers to a solid form of a compound wherein theconstituent molecules are packed in a regularly ordered, repeatingpattern. A crystalline form can include triclinic, monoclinic,orthorhombic, tetragonal, trigonal, hexagonal, and cubic crystalgeometries. A crystalline form can include one or more regions, i.e.,grains, with distinct crystal boundaries. A crystalline solid caninclude two or more crystal geometries.

“Amorphous form” refers to a solid form of a compound having no definitecrystal structure, i.e., lacking a regularly ordered, repeating patternof constituent molecules.

“Isolating” refers to the process of isolating at least a portion of afirst substance (e.g., a precipitate) from a mixture including thesubstance and at least one additional substance. In some instances, theisolated substance is substantially free of at least one of theadditional substances present in the original mixture.

“Substantially free” refers to an amount of 10% or less of another formor impurity, preferably 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less of anotherform or impurity. Preferably, substantially free refers to a crystallineform of a compound of Formula (I) that contains less than 5% of othercrystalline or amorphous forms of a compound of Formula (I). Preferably,substantially free refers to a crystalline form of a compound of Formula(I) that contains less than 1% of other crystalline or amorphous formsof a compound of Formula (I).

“About” means a range of values including the specified value, which aperson of ordinary skill in the art would consider reasonably similar tothe specified value. In some embodiments, the term “about” means withina standard deviation using measurements generally acceptable in the art.In some embodiments, “about” means a range extending to +1-10% of thespecified value. In some embodiments, “about” means a range extending to+1-5% of the specified value. In some embodiments, “about” means a rangeextending to +1-2% of the specified value. In some embodiments, “about”means the specified value.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present disclosurecontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of salts derived frompharmaceutically-acceptable inorganic bases include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. When compounds of the presentdisclosure contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentdisclosure contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. Unnatural proportions of an isotope may bedefined as ranging from the amount found in nature to an amountconsisting of 100% of the atom in question. For example, the compoundsmay incorporate radioactive isotopes, such as for example tritium (³H),or carbon-14 (¹⁴C), or non-radioactive isotopes, such as deuterium (²H)or carbon-13 (¹³C). Such isotopic of the compounds of the disclosure mayfind additional utility, including but not limited to, as diagnosticand/or imaging reagents, or as cytotoxic/radiotoxic therapeutic agents.Additionally, isotopic variants of the compounds of the disclosure canhave altered pharmacokinetic and pharmacodynamic characteristics whichcan contribute to enhanced safety, tolerability or efficacy duringtreatment. All isotopic variations of the compounds of the presentinventiondisclosure, whether radioactive or not, are intended to beencompassed within the scope of the present disclosure.

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering an inhibitor of CD73 or apharmaceutical composition comprising same) initiated after a disease,disorder or condition, or a symptom thereof, has been diagnosed,observed, and the like so as to eliminate, reduce, suppress, mitigate,or ameliorate, either temporarily or permanently, at least one of theunderlying causes of a disease, disorder, or condition afflicting asubject, or at least one of the symptoms associated with a disease,disorder, condition afflicting a subject. Thus, treatment includesinhibiting (e.g., arresting the development or further development ofthe disease, disorder or condition or clinical symptoms associationtherewith) an active disease.

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering an CD73 inhibitor or apharmaceutical composition comprising same) initiated in a manner (e.g.,prior to the onset of a disease, disorder, condition or symptom thereof)so as to prevent, suppress, inhibit or reduce, either temporarily orpermanently, a subject's risk of developing a disease, disorder,condition or the like (as determined by, for example, the absence ofclinical symptoms) or delaying the onset thereof, generally in thecontext of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount capable of having any detectable, positiveeffect on any symptom, aspect, or characteristic of a disease, disorderor condition when administered to the subject. The therapeuticallyeffective amount can be ascertained by measuring relevant physiologicaleffects, and it can be adjusted in connection with the dosing regimenand diagnostic analysis of the subject's condition, and the like. By wayof example, measurement of the serum level of an CD73 inhibitor (or,e.g., a metabolite thereof) at a particular time post-administration maybe indicative of whether a therapeutically effective amount has beenused.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., serumconcentration) or subjective parameter (e.g., a subject's feeling ofwell-being).

“Substantially pure” indicates that a component makes up greater thanabout 50% of the total content of the composition, and typically greaterthan about 60% of the total content. More typically, “substantiallypure” refers to compositions in which at least 75%, at least 85%, atleast 90% or more of the total composition is the component of interest.In some cases, the component of interest will make up greater than about90%, or greater than about 95% of the total content of the composition.

As used herein, the terms “CD73 inhibitor”, “CD73 blocker”, “adenosineby 5′-nucleotidase, ecto inhibitor”, “NT5E inhibitor”, “5NT inhibitor”and all other related art-accepted terms refer to a compound capable ofmodulating, either directly or indirectly, the CD73 receptor in an invitro assay, an in vivo model, and/or other means indicative oftherapeutic efficacy. The terms also refer to compounds that exhibit atleast some therapeutic benefit in a human subject. An CD73 inhibitor maybe a competitive, noncompetitive, or irreversible CD73 inhibitor. “Acompetitive CD73 inhibitor” is a compound that reversibly inhibits CD73enzyme activity at the catalytic site; “a noncompetitive CD73 inhibitor”is a compound that reversibly inhibits CD73 enzyme activity at anon-catalytic site; and “an irreversible CD73 inhibitor” is a compoundthat irreversibly eliminates CD73 enzyme activity by forming a covalentbond (or other stable means of inhibiting enzyme function) with theenzyme.

III. Crystalline Forms

The present disclosure provides crystalline forms of(((((2R,3S,4R,5R)-5-(6-chloro-4-(((S)-1-(2-fluorophenyl)ethyl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonicacid: represented by Formula (I), including solvate and hydrate forms.

In one aspect, the present disclosure provides a crystalline form of acompound having Formula (I):

wherein the crystalline form is any one of crystalline Forms I to VI,each of which is characterized by an X-ray powder diffraction (XRPD)pattern as described herein.

Methods for collection of XRPD data are known in the art, and any suchmethods can be used for characterizing the crystalline forms of thecompound of formula (I). For example, the X-ray powder diffractionpatterns described herein can be generated using Cu Kα1 radiation.

In some embodiments, the crystalline form described herein is furthercharacterized by a differential scanning calorimetry (DSC) thermogram.

In some embodiments, the crystalline form described herein is furthercharacterized by a Nuclear Magnetic Resonance spectrum, such as a ¹H NMRspectrum.

In some embodiments, the crystallize form described herein is furthercharacterized by a dynamic vapor sorption (DVS) isotherm.

III-1. Crystalline Form I

In one embodiment, the present disclosure provides crystalline Form I ofa compound of Formula (I), characterized by an X-ray powder diffraction(XRPD) pattern including three or more peaks at 11.1, 11.6, 13.8, 14.7,15.4, 16.6, 17.0, 18.6, 19.3, 20.1, 21.3, 22.1, 23.0, 24.8, 26.6, 27.3,and 29.1 degrees 2θ (±0.2 degrees 2θ).

Crystalline Form I of the compound of Formula (I) can be characterizedby an X-ray powder diffraction (XRPD) pattern having one or more, e.g.,two, three, four, five, seven, or more, peaks at 11.1, 11.6, 13.8, 14.7,15.4, 16.6, 17.0, 18.6, 19.3, 20.1, 21.3, 22.1, 23.0, 24.8, 26.6, 27.3,and 29.1 degrees 2θ (±0.2 degrees 2θ), wherein the XRPD is made usingCuK_(α1) radiation. In some embodiments, the crystalline Form I ischaracterized by an XRPD pattern including four or more peaks at 11.1,11.6, 13.8, 14.7, 15.4, 16.6, 17.0, 18.6, 19.3, 20.1, 21.3, 22.1, 23.0,24.8, 26.6, 27.3, and 29.1 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the crystalline Form I is characterized by an XRPD patternincluding five or more peaks at 11.1, 11.6, 13.8, 14.78, 15.4, 16.6,17.0, 18.6, 19.3, 20.1, 21.3, 22.1, 23.0, 24.8, 26.6, 27.3, and 29.1degrees 2θ (±0.2 degrees 2θ). In some embodiments, the crystalline FormI is characterized by an XRPD pattern including six or more peaks at11.1, 11.6, 13.8, 14.78, 15.4, 16.6, 17.0, 18.6, 19.3, 20.1, 21.3, 22.1,23.0, 24.8, 26.6, 27.3, and 29.1 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the crystalline Form I is characterized by an XRPD patternincluding seven or more peaks at 11.1, 11.6, 13.8, 14.78, 15.4, 16.6,17.0, 18.6, 19.3, 20.1, 21.3, 22.1, 23.0, 24.8, 26.6, 27.3, and 29.1degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form I is characterized by an XRPDpattern including peaks at 11.1, 13.8, 18.6, 20.1, 23.0, and 24.8degrees 2θ (±0.2 degrees 2θ). In some embodiments, the XRPD patternfurther includes one or more peaks at 11.6, 14.7, 15.4, 16.6, 17.0,19.3, 21.3, 22.1, 24.8, 26.6, 27.3, and 29.1 degrees 2θ (±0.2 degrees2θ). In some embodiments, the XRPD pattern further includes two or morepeaks at 11.6, 14.7, 15.4, 16.6, 17.0, 19.3, 21.3, 22.1, 24.8, 26.6,27.3, and 29.1 degrees 2θ (±0.2 degrees 2θ). In some embodiments, theXRPD pattern further includes three or more peaks at 11.6, 14.7, 15.4,16.6, 17.0, 19.3, 21.3, 22.1, 24.8, 26.6, 27.3, and 29.1 degrees 2θ(±0.2 degrees 2θ). In some embodiments, the XRPD pattern furtherincludes four or more peaks at 11.6, 14.7, 15.4, 16.6, 17.0, 19.3, 21.3,22.1, 24.8, 26.6, 27.3, and 29.1 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes five or more peaks at11.6, 14.7, 15.4, 16.6, 17.0, 19.3, 21.3, 22.1, 24.8, 26.6, 27.3, and29.1 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form I is characterized by an XRPDpattern including peaks at 11.1, 11.6, 13.8, 14.7, 15.4, 16.6, 17.0,18.6, 19.3, 20.1, 21.3, 22.1, 23.0, 24.8, 26.6, 27.3, and 29.1 degrees2θ (±0.2 degrees 2θ). In some embodiments, the crystalline Form I ischaracterized by an XRPD pattern including peaks at 6.3, 8.0, 9.3, 11.1,11.6, 13.8, 14.7, 15.4, 16.6, 17.0, 18.6, 19.3, 20.1, 21.3, 22.1, 23.0,24.8, 26.6, 27.3, 29.1, 31.0, 31.9, and 33.4 degrees 28 (±0.2 degrees2θ).

In some embodiments, the crystalline Form I is characterized by an XRPDpattern substantially in accordance with FIG. 1.

In some embodiments, crystalline Form I is substantially free of othercrystalline or amorphous forms of the compound of Formula (I).

In some embodiments, the crystalline Form I is characterized by adifferential scanning calorimetry (DSC) thermogram including anendotherm at from about 155° C. to about 167° C. In some embodiments,the crystalline Form I is further characterized by a differentialscanning calorimetry (DSC) thermogram including an endothermic peak atabout 163.9° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram further includes an exotherm at from about167 to about 210° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram is characterized by an exothermic peak atabout 176.1° C. In some embodiments, the crystalline Form I is furthercharacterized by a melting point of about 163.9° C. as determined by adifferential scanning calorimetry thermogram (DSC). In some embodiments,the crystalline Form I is further characterized by a melting point onsetof about 155.1° C. as determined by a differential scanning calorimetrythermogram (DSC).

In some embodiments, the crystalline Form I is further characterized bya differential scanning calorimetry (DSC) thermogram substantially inaccordance with FIG. 2.

In some embodiments, the crystalline Form I is characterized by an XRPDpattern substantially in accordance with FIG. 1; and is furthercharacterized by a differential scanning calorimetry (DSC) thermogramsubstantially in accordance with FIG. 2.

A ¹H NMR spectrum of the crystalline Form I can be used to determine thecontent of one or more residual solvents (e.g., ethanol and/or toluene).In some embodiments, the crystalline Form I has ethanol in an amount of0.04% by weight, as determined by a ¹H NMR spectrum. In someembodiments, the crystalline Form I is substantially free of toluene, asdetermined by a ¹H NMR spectrum.

In some embodiments, the crystalline Form I is characterized by a DVSisotherm characterized by a change in mass of between 0.5% and 6.5% atbetween 40% RH and 70% RH. In some embodiments, the crystalline Form Iis characterized by a DVS isotherm characterized by a change in mass ofbetween 2% and 5% between 50% RH and 60% RH. In some embodiments, thecrystalline Form I is characterized by a DVS isotherm substantially inaccordance with FIG. 16.

III-2. Crystalline Form II

In one embodiment, the present disclosure provides crystalline Form IIof a compound of Formula (I), characterized by an X-ray powderdiffraction (XRPD) pattern including three or more peaks at 10.1, 10.8,12.8, 13.7, 16.5, 17.7, 19.0, 22.8, and 24.6 degrees 2θ (±0.2 degrees2θ).

Crystalline Form II of the compound of Formula (I) can be characterizedby an X-ray powder diffraction (XRPD) pattern having one or more, e.g.,two, three, four, five, seven, or more, peaks at 10.1, 10.8, 12.8, 13.7,16.5, 17.7, 19.0, 22.8, and 24.6 degrees 28 (±0.2 degrees 28), whereinthe XRPD is made using CuK_(α1) radiation. In some embodiments, thecrystalline Form II of the compound of Formula (I) is characterized byan XRPD pattern including four or more peaks at 10.1, 10.8, 12.8, 13.7,16.5, 17.7, 19.0, 22.8, and 24.6 degrees 2θ (±0.2 degrees 28). In someembodiments, the crystalline Form II of the compound of Formula (I) ischaracterized by an XRPD pattern including five or more peaks at 10.1,10.8, 12.8, 13.7, 16.5, 17.7, 19.0, 22.8, and 24.6 degrees 2θ (±0.2degrees 2θ). In some embodiments, the crystalline Form II of thecompound of Formula (I) is characterized by an XRPD pattern includingsix or more peaks at 10.1, 10.8, 12.8, 13.7, 16.5, 17.7, 19.0, 22.8, and24.6 degrees 2θ (±0.2 degrees 2θ). In some embodiments, the crystallineForm II of the compound of Formula (I) is characterized by an XRPDpattern including seven or more peaks at 10.1, 10.8, 12.8, 13.7, 16.5,17.7, 19.0, 22.8, and 24.6 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form II of the compound of Formula(I) is characterized by an XRPD pattern including a peak at 16.5 degrees2θ (±0.2 degrees 2θ). In some embodiments, the crystalline Form II ofthe compound of Formula (I) is characterized by an XRPD patternincluding peaks at 16.5, 22.8, and 24.6 degrees 2θ (±0.2 degrees 2θ). Insome embodiments, the XRPD pattern further includes one or more peaks at10.1, 10.8, 12.8, 13.7, 17.7, and 19.0 degrees 2θ (±0.2 degrees 2θ). Insome embodiments, the XRPD pattern further includes two or more peaks at10.1, 10.8, 12.8, 13.7, 17.7, and 19.0 degrees 2θ (±0.2 degrees 2θ). Insome embodiments, the XRPD pattern further includes three or more peaksat 10.1, 10.8, 12.8, 13.7, 17.7, and 19.0 degrees 2θ (±0.2 degrees 2θ).In some embodiments, the XRPD pattern further includes four or morepeaks at 10.1, 10.8, 12.8, 13.7, 17.7, and 19.0 degrees 2θ (±0.2 degrees2θ). In some embodiments, the XRPD pattern further includes five or morepeaks at 10.1, 10.8, 12.8, 13.7, 17.7, and 19.0 degrees 2θ (±0.2 degrees2θ).

In some embodiments, the crystalline Form II is characterized by an XRPDpattern including peaks at 10.1, 10.8, 12.8, 13.7, 16.5, 17.7, 19.0,22.8, and 24.6 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form II is characterized by an XRPDpattern substantially in accordance with FIG. 3.

In some embodiments, the crystalline Form II is substantially free ofother crystalline or amorphous forms of a compound of Formula (I).

In some embodiments, the crystalline Form II is characterized by adifferential scanning calorimetry (DSC) thermogram including anendotherm at from about 156° C. to about 171° C. In some embodiments,the crystalline Form II is further characterized by a differentialscanning calorimetry (DSC) thermogram including an endothermic peak atabout 166.5° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram further includes an exotherm at from about170° C. to about 210° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram is characterized by an exothermic peak atabout 179.3° C. In some embodiments, the crystalline Form II is furthercharacterized by a melting point of about 166.5° C. as determined by adifferential scanning calorimetry thermogram (DSC). In some embodiments,the crystalline Form II is further characterized by a melting pointonset of about 157.4° C. as determined by a differential scanningcalorimetry thermogram (DSC).

In some embodiments, the crystalline Form II is further characterized bya differential scanning calorimetry (DSC) thermogram substantially inaccordance with FIG. 4.

In some embodiments, the crystalline Form II is characterized by an XRPDpattern substantially in accordance with FIG. 3; and is furthercharacterized by a differential scanning calorimetry (DSC) thermogramsubstantially in accordance with FIG. 4.

A ¹H NMR spectrum of the crystalline Form II can be used to determinethe content of one or more residual solvents (e.g., ethanol). In someembodiments, the crystalline Form II has ethanol in an amount of 0.68%by weight, as determined by a ¹H NMR spectrum.

In some embodiments, the crystalline Form II is characterized by a DVSisotherm characterized by a change in mass of between 1% and 4% atbetween 40% RH and 70% RH. In some embodiments, the crystalline Form IIis characterized by a DVS isotherm characterized by a change in mass ofbetween 1.5% and 3.5% between 40% RH and 70% RH. In some embodiments,the crystalline Form II is characterized by a DVS isotherm substantiallyin accordance with FIG. 17.

III-3. Crystalline Form III

In one embodiment, the present disclosure provides crystalline Form IIIof a compound of Formula (I), characterized by an X-ray powderdiffraction (XRPD) pattern including three or more peaks at 6.6, 10.9,14.2, 16.1, 18.4, 19.3, 20.2, 22.0, 24.7, and 28.1 degrees 2θ (±0.2degrees 2θ).

Crystalline Form III of the compound of Formula (I) can be characterizedby an X-ray powder diffraction (XRPD) pattern having one or more, e.g.,two, three, four, five, or more, peaks at 6.6, 10.9, 14.2, 16.1, 18.4,19.3, 20.2, 22.0, 24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ), whereinthe XRPD is made using CuK_(α1) radiation. In some embodiments, thecrystalline Form III of the compound of Formula (I) is characterized byan XRPD pattern including four or more peaks at 6.6, 10.9, 14.2, 16.1,18.4, 19.3, 20.2, 22.0, 24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ). Insome embodiments, the crystalline Form III of the compound of Formula(I) is characterized by an XRPD pattern including five or more peaks at6.6, 10.9, 14.2, 16.1, 18.4, 19.3, 20.2, 22.0, 24.7, and 28.1 degrees 2θ(±0.2 degrees 2θ). In some embodiments, the crystalline Form III of thecompound of Formula (I) is characterized by an XRPD pattern includingsix or more peaks at 6.6, 10.9, 14.2, 16.1, 18.4, 19.3, 20.2, 22.0,24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ). In some embodiments, thecrystalline Form III of the compound of Formula (I) is characterized byan XRPD pattern including seven or more peaks at 6.6, 10.9, 14.2, 16.1,18.4, 19.3, 20.2, 22.0, 24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form III of the compound of Formula(I) is characterized by an XRPD pattern including peaks at 6.6, 10.9,14.2, 16.1, 18.4, and 19.3 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes one or more peaks at20.2, 22.0, 24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes two or more peaks at20.2, 22.0, 24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes three or more peaks at20.2, 22.0, 24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes four peaks at 20.2, 22.0,24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ). In some embodiments, theXRPD pattern further includes peaks at 29.7, 32.0, and 33.5 degrees 2θ(±0.2 degrees 2θ).

In some embodiments, the crystalline Form III is characterized by anXRPD pattern including peaks at 6.6, 10.9, 14.2, 16.1, 18.4, 19.3, 20.2,22.0, 24.7, and 28.1 degrees 2θ (±0.2 degrees 2θ). In some embodiments,the crystalline Form III is characterized by an XRPD pattern includingpeaks at 6.6, 10.9, 14.2, 16.1, 18.4, 19.3, 20.2, 22.0, 24.7, 28.1,29.7, 32.0, and 33.5 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form III is characterized by anXRPD pattern substantially in accordance with FIG. 5.

In some embodiments, the crystalline Form III is substantially free ofother crystalline or amorphous forms of a compound of Formula (I).

In some embodiments, the crystalline Form III is characterized by adifferential scanning calorimetry (DSC) thermogram including anendotherm at from about 149° C. to about 183° C. In some embodiments,the crystalline Form III is further characterized by a differentialscanning calorimetry (DSC) thermogram including an endothermic peak atabout 161.8° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram further includes an exotherm at from about183 to about 210° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram is characterized by an exothermic peak atabout 188.2° C. In some embodiments, the crystalline Form III is furthercharacterized by a melting point of about 161.8° C. as determined by adifferential scanning calorimetry thermogram (DSC). In some embodiments,the crystalline Form III is further characterized by a melting pointonset of about 149.6° C. as determined by a differential scanningcalorimetry thermogram (DSC).

In some embodiments, the crystalline Form III is further characterizedby a differential scanning calorimetry (DSC) thermogram substantially inaccordance with FIG. 6.

In some embodiments, the crystalline Form III is characterized by anXRPD pattern substantially in accordance with FIG. 5; and is furthercharacterized by a differential scanning calorimetry (DSC) thermogramsubstantially in accordance with FIG. 6.

A ¹H NMR spectrum of the crystalline Form III can be used to determinethe content of one or more residual solvents (e.g., ethanol and/ormethyl tert-butyl ether). In some embodiments, the crystalline Form IIIis substantially free of ethanol, as determined by a ¹H NMR spectrum. Insome embodiments, the crystalline Form III has methyl tert-butyl etherin an amount of 0.40% by weight, as determined by a ¹H NMR spectrum.

III-4. Crystalline Form IV

In one embodiment, the present disclosure provides crystalline Form IVof a compound of Formula (I), characterized by an X-ray powderdiffraction (XRPD) pattern including three or more peaks at 6.0, 11.2,14.1, 17.0, 19.5, 23.2, 25.1, 27.1, and 28.9 degrees 2θ (±0.2 degrees2θ).

Crystalline Form IV of the compound of Formula (I) can be characterizedby an X-ray powder diffraction (XRPD) pattern having one or more, e.g.,two, three, four, five, or more, peaks at 6.0, 11.2, 14.1, 17.0, 19.5,23.2, 25.1, 27.1, and 28.9 degrees 2θ (±0.2 degrees 2θ), wherein theXRPD is made using CuK_(α1) radiation. In some embodiments, thecrystalline Form IV of the compound of Formula (I) is characterized byan XRPD pattern including four or more peaks at 6.0, 11.2, 14.1, 17.0,19.5, 23.2, 25.1, 27.1, and 28.9 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the crystalline Form IV of the compound of Formula (I) ischaracterized by an XRPD pattern including five or more peaks at 6.0,11.2, 14.1, 17.0, 19.5, 23.2, 25.1, 27.1, and 28.9 degrees 2θ (±0.2degrees 2θ). In some embodiments, the crystalline Form IV of thecompound of Formula (I) is characterized by an XRPD pattern includingsix or more peaks at 6.0, 11.2, 14.1, 17.0, 19.5, 23.2, 25.1, 27.1, and28.9 degrees 2θ (±0.2 degrees 2θ). In some embodiments, the crystallineForm IV of the compound of Formula (I) is characterized by an XRPDpattern including seven or more peaks at 6.0, 11.2, 14.1, 17.0, 19.5,23.2, 25.1, 27.1, and 28.9 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form IV of the compound of Formula(I) is characterized by an XRPD pattern including peaks at 14.1, 17.0,19.5, 23.2, and 25.1 degrees 2θ (±0.2 degrees 2θ). In some embodiments,the XRPD pattern further includes one or more peaks at 6.0, 11.2, 27.1,and 28.9 degrees 2θ (±0.2 degrees 2θ). In some embodiments, the XRPDpattern further includes two or more peaks at 6.0, 11.2, 27.1, and 28.9degrees 2θ (±0.2 degrees 2θ). In some embodiments, the XRPD patternfurther includes three or more peaks at 6.0, 11.2, 27.1, and 28.9degrees 2θ (±0.2 degrees 2θ). In some embodiments, the XRPD patternfurther includes four peaks at 6.0, 11.2, 27.1, and 28.9 degrees 2θ(±0.2 degrees 2θ).

In some embodiments, the crystalline Form IV is characterized by an XRPDpattern including peaks at 6.0, 11.2, 14.1, 17.0, 19.5, 23.2, 25.1,27.1, and 28.9 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form IV is characterized by an XRPDpattern substantially in accordance with FIG. 7.

In some embodiments, the crystalline Form IV is substantially free ofother crystalline or amorphous forms of a compound of Formula (I).

A ¹H NMR spectrum of the crystalline Form IV can be used to determinethe content of one or more residual solvents (e.g., THF and/or ethylacetate). In some embodiments, the crystalline Form IV has a content ofTHF in an amount of 0.05%, as determined by a ¹H NMR spectrum. In someembodiments, the crystalline Form IV has ethyl acetate in an amount of0.38% by weight, as determined by a ¹H NMR spectrum.

III-5. Crystalline Form V

In one embodiment, the present disclosure provides crystalline Form V ofa compound of Formula (I), characterized by an X-ray powder diffraction(XRPD) pattern including three or more peaks at 10.4, 15.1, 15.8, 16.3,16.8, 18.5, 19.1, 19.7, 21.7, 22.1, 23.0, 23.5, 26.0, 26.5, 28.4, 28.9,and 31.4 degrees 2θ (±0.2 degrees 2θ).

Crystalline Form V of the compound of Formula (I) can be characterizedby an X-ray powder diffraction (XRPD) pattern having one or more, e.g.,two, three, four, five, or more, peaks at 10.4, 15.1, 15.8, 16.3, 16.8,18.5, 19.1, 19.7, 21.7, 22.1, 23.0, 23.6, 26.0, 26.5, 28.4, 28.9, and31.4 degrees 2θ (±0.2 degrees 2θ), wherein the XRPD is made usingCuK_(α1) radiation. In some embodiments, the crystalline Form V of thecompound of Formula (I) is characterized by an XRPD pattern includingfour or more peaks at 10.4, 15.1, 15.8, 16.3, 16.8, 18.5, 19.1, 19.7,21.7, 22.1, 23.0, 23.6, 26.0, 26.5, 28.4, 28.9, and 31.4 degrees 2θ(±0.2 degrees 2θ). In some embodiments, the crystalline Form V of thecompound of Formula (I) is characterized by an XRPD pattern includingfive or more peaks at 10.4, 15.1, 15.8, 16.3, 16.8, 18.5, 19.1, 19.7,21.7, 22.1, 23.0, 23.6, 26.0, 26.5, 28.4, 28.9, and 31.4 degrees 2θ(±0.2 degrees 2θ). In some embodiments, the crystalline Form V of thecompound of Formula (I) is characterized by an XRPD pattern includingsix or more peaks at 10.4, 15.1, 15.8, 16.3, 16.8, 18.5, 19.1, 19.7,21.7, 22.1, 23.0, 23.6, 26.0, 26.5, 28.4, 28.9, and 31.4 degrees 2θ(±0.2 degrees 2θ). In some embodiments, the crystalline Form V of thecompound of Formula (I) is characterized by an XRPD pattern includingseven or more peaks at 10.4, 15.1, 15.8, 16.3, 16.8, 18.5, 19.1, 19.7,21.7, 22.1, 23.0, 23.6, 26.0, 26.5, 28.4, 28.9, and 31.4 degrees 2θ(±0.2 degrees 2θ).

In some embodiments, the crystalline Form V of the compound of Formula(I) is characterized by an XRPD pattern including peaks at 15.8, 16.3,16.8, 18.5, 19.1, 21.7, 22.1, and 23.0 degrees 2θ (±0.2 degrees 2θ). Insome embodiments, the XRPD pattern further includes one or more peaks at10.4, 15.1, 19.7, and 23.6 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes two or more peaks at10.4, 15.1, 19.7, 23.6 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes three or more peaks at10.4, 15.1, 19.7, 23.6 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the XRPD pattern further includes four peaks at 10.4, 15.1,19.7, 23.6 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form V is characterized by an XRPDpattern including peaks at 10.4, 15.1, 15.8, 16.3, 16.8, 18.5, 19.1,19.7, 21.7, 22.1, 23.0, and 23.6 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form V is characterized by an XRPDpattern substantially in accordance with FIG. 8.

In some embodiments, the crystalline Form V is substantially free ofother crystalline or amorphous forms of a compound of Formula (I).

In some embodiments, the crystalline Form V is characterized by adifferential scanning calorimetry (DSC) thermogram including anendotherm at from about 135° C. to about 172° C. In some embodiments,the crystalline Form V is further characterized by a differentialscanning calorimetry (DSC) thermogram including an endothermic peak atabout 150.4° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram further includes an exotherm at from about171 to about 210° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram is characterized by an exothermic peak atabout 177.8° C. In some embodiments, the crystalline Form V is furthercharacterized by a melting point of about 150.4° C. as determined by adifferential scanning calorimetry thermogram (DSC). In some embodiments,the crystalline Form V is further characterized by a melting point onsetof about 135.8° C. as determined by a differential scanning calorimetrythermogram (DSC).

In some embodiments, the crystalline Form V is further characterized bya differential scanning calorimetry (DSC) thermogram substantially inaccordance with FIG. 9.

In some embodiments, the crystalline Form V is characterized by an XRPDpattern substantially in accordance with FIG. 8; and is furthercharacterized by a differential scanning calorimetry (DSC) thermogramsubstantially in accordance with FIG. 9.

A ¹H NMR spectrum of the crystalline Form V can be used to determine thecontent of one or more residual solvents (e.g., ethanol and/or ethylacetate). In some embodiments, the crystalline Form V has a content ofethanol in an amount of 7.6%, as determined by a ¹H NMR spectrum. Insome embodiments, the crystalline Form V is in an ethyl acetate solvate,an ethanol solvate form, or a combination thereof. In some embodiments,crystalline Form V is an ethanol solvate.

III-5. Crystalline Form VI

In one embodiment, the present disclosure provides crystalline Form VIof a compound of Formula (I), characterized by an X-ray powderdiffraction (XRPD) pattern including three or more peaks at 5.8, 10.4,16.2, 19.4, 21.3, 22.4, 24.4, 27.5, and 31.1 degrees 2θ (±0.2 degrees2θ).

Crystalline Form VI of the compound of Formula (I) can be characterizedby an X-ray powder diffraction (XRPD) pattern having one or more, e.g.,two, three, four, five, or more, peaks at 5.8, 10.4, 16.2, 19.4, 21.3,22.4, 24.4, 27.5, and 31.1 degrees 2θ (±0.2 degrees 2θ), wherein theXRPD is made using CuK_(α1) radiation. In some embodiments, thecrystalline Form VI of the compound of Formula (I) is characterized byan XRPD pattern including four or more peaks at 5.8, 10.4, 16.2, 19.4,21.3, 22.4, 24.4, 27.5, and 31.1 degrees 2θ (±0.2 degrees 2θ). In someembodiments, the crystalline Form VI of the compound of Formula (I) ischaracterized by an XRPD pattern including five or more peaks at 5.8,10.4, 16.2, 19.4, 21.3, 22.4, 24.4, 27.5, and 31.1 degrees 2θ (±0.2degrees 2θ). In some embodiments, the crystalline Form VI of thecompound of Formula (I) is characterized by an XRPD pattern includingsix or more peaks at 5.8, 10.4, 16.2, 19.4, 21.3, 22.4, 24.4, 27.5, and31.1 degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form VI of the compound of Formula(I) is characterized by an XRPD pattern including peaks at 19.4, 21.3,22.4, and 24.4 degrees 2θ (±0.2 degrees 2θ). In some embodiments, theXRPD pattern further includes one or more peaks at 5.8, 10.4, 27.5 and31.1 degrees 2θ (±0.2 degrees 2θ). In some embodiments, the XRPD patternfurther includes two or more peaks at 5.8, 10.4, 27.5 and 31.1 degrees2θ (±0.2 degrees 2θ). In some embodiments, the XRPD pattern furtherincludes three or more peaks at 5.8, 10.4, 27.5 and 31.1 degrees 2θ(±0.2 degrees 2θ).

In some embodiments, the crystalline Form VI is characterized by an XRPDpattern including peaks at 5.8, 10.4, 16.2, 19.4, 21.3, 22.4, and 24.4degrees 2θ (±0.2 degrees 2θ).

In some embodiments, the crystalline Form VI is characterized by an XRPDpattern substantially in accordance with FIG. 10.

In some embodiments, the crystalline Form VI is substantially free ofother crystalline or amorphous forms of a compound of Formula (I).

In some embodiments, the crystalline Form VI is characterized by adifferential scanning calorimetry (DSC) thermogram including anendotherm at from about 116° C. to about 170° C. In some embodiments,the crystalline Form VI is further characterized by a differentialscanning calorimetry (DSC) thermogram including an endothermic peak atabout 142.9° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram further includes an exotherm at from about187 to about 210° C. In some embodiments, the differential scanningcalorimetry (DSC) thermogram is characterized by an exothermic peak atabout 193.9° C. In some embodiments, the crystalline Form VI is furthercharacterized by a melting point of about 142.9° C. as determined by adifferential scanning calorimetry thermogram (DSC). In some embodiments,the crystalline Form VI is further characterized by a melting pointonset of about 116.6° C. as determined by a differential scanningcalorimetry thermogram (DSC).

In some embodiments, the crystalline Form VI is further characterized bya differential scanning calorimetry (DSC) thermogram substantially inaccordance with FIG. 11.

In some embodiments, the crystalline Form VI is characterized by an XRPDpattern substantially in accordance with FIG. 10; and is furthercharacterized by a differential scanning calorimetry (DSC) thermogramsubstantially in accordance with FIG. 11.

IV. Processes for Preparing Crystalline Forms

In another aspect, the present disclosure provides a process forpreparing a crystalline form of a compound of Formula (I). The processincludes:

-   -   a) forming a first mixture including a compound of Formula (I)        and a solvent at a temperature of at least 20° C.; and    -   b) adding an anti-solvent to the first mixture to form a second        mixture, or    -   a) forming a first mixture including a compound of Formula (I)        and a solvent at a temperature of at least 20° C.;    -   c) cooling the first or second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline form of        Formula (I),    -   wherein the solvent is a C₁₋₄alkyl alcohol, a di-(C₁₋₄alkyl)        ether, a 5-6 membered cyclic ether, acetic acid, or water; and        the anti-solvent is a C₅₋₇ alkane, C₁₋₄alkyl alcohol, a        di-(C₁₋₄alkyl) ether,    -   a 5-6 membered cyclic ether, a di-(C₁₋₄alkyl) ketone,        C₁₋₄alkyl-C(O)O—C₁₋₄alkyl, or an aromatic hydrocarbon solvent,    -   provided that the solvent and anti-solvent are not each a        C₁₋₄alkyl alcohol, a di-(C₁₋₄alkyl) ether, or a 5-6 membered        cyclic ether.

In general, the morphology of the starting material (i.e., the compoundof Formula (I)) is unimportant with respect to the successful recoveryof crystalline material, although the kinetics of initial dissolutionmay be affected and a greater proportion of solvent may be required. Forexample, either amorphous material obtained via lyophilization orpreexisting crystalline material may be used to obtain the desiredcrystalline form. In some cases, it may be beneficial to first isolatethe compound of Formula (I) as a first crystalline form, and use thefirst crystalline form as the starting material to obtain the desiredcrystalline form. In special cases, a metastable form is heated undervacuum to synthesize the desired form.

The sodium content of the starting material can also affect the successof the crystallization. In general, samples with a sodium content of0.5% by weight or greater are more difficult to crystallize, althoughmore solvent can be used to help mitigate this problem.

Single Solvents and Binary Solvent Mixtures

Several solvents can be used to generate the desired crystalline form,either through use of a single solvent or a binary solvent mixture. Inthe case of a single solvent the starting material can be dissolved byheating in a solvent capable of forming a reasonably concentratedsolution, followed by cooling to precipitate the desired crystallineform. Suitable solvents for use alone or as a mixture include but arenot limited to isopropanol, ethanol, methanol, acetonitrile, aceticacid, tetrahydrofuran, and water. Alternatively, the starting materialcan be dissolved in a solvent capable of forming a reasonablyconcentrated solution at ambient temperature, followed by slowevaporation of the solvent (e.g., under ambient conditions, under a flowof an inert gas (i.e., N₂ or Ar) or under reduced pressure), toprecipitate the desired crystalline form.

In the case of a binary solvent mixture, the material is first dissolvedin a solvent capable of forming a reasonably concentrated solution asoutlined above followed by the addition of a less polar solvent (e.g.,an anti-solvent) in which the material is not readily soluble toprecipitate the desired material. In cases wherein the solvent wasinitially heated to dissolve the material, the anti-solvent can be addedwhile the solution is hot, or after it has cooled, e.g., to roomtemperature. In a selected example, the material is dissolved in ethanolat room temperature and ethyl acetate added to precipitate the desiredcrystalline form. Suitable precipitating solvents (as anti-solvents)include but are not limited to toluene, ethyl acetate, diethyl ether,acetone, methyl tert-butyl ether, isopropanol, pentane, hexane, heptane,and acetonitrile.

In some embodiments, the solvent is a C₁₋₄alkyl alcohol, adi-(C₁₋₄alkyl) ether, or a 5-6 membered cyclic ether, or a mixturethereof. In some embodiments, the solvent is a C₁₋₄alkyl alcohol or a5-6 membered cyclic ether. Suitable C₁₋₄alkyl alcohols include, but arenot limited to, methanol, ethanol, or iso-propanol. Suitabledi-(C₁₋₄alkyl) ethers include, but are not limited to, diethyl ether,methyl ethyl ether, or methyl tert-butyl ether. Suitable 5-6 memberedcyclic ethers include, but are not limited to, tetrahydrofuran, methyltetrahydrofuran, or dioxanes. In some embodiments, the solvent isethanol, tetrahydrofuran or a mixture thereof. In some embodiments, thesolvent is ethanol. In some embodiments, the solvent is tetrahydrofuran.In some embodiments, the solvent further comprises water.

In some embodiments, the present disclosure provides a process forpreparing the crystalline Form II, V or VI of the compound of Formula(I), the process including:

-   -   a) forming a first mixture including a compound of Formula (I)        and ethanol at a temperature of at least 20° C.;    -   c) cooling the first mixture and stirring to form a precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form II or        V of Formula (I).

The temperature at which step a) is conducted can influence the identityof the resulting crystalline form. Without wishing to be bound bytheory, low temperatures favor the formation of crystalline Form VI,moderate temperatures favor the formation of crystalline Form V, andhigher temperatures favor the formation of crystalline Form II. In someembodiments, step a) is conducted at a temperature from 20° C. to 30° C.In some embodiments, step a) is conducted at a temperature from 30° C.to 40° C. In some embodiments, step a) is conducted at a temperaturefrom 40° C. to 65° C.

In some embodiments, the present disclosure provides a process forpreparing crystalline Form I, II, III, IV or V of the compound ofFormula (I), the process including:

-   -   a) forming a first mixture including a compound of Formula (I)        and a solvent at a temperature of at least 20° C.;    -   b) adding an anti-solvent to the first mixture to form a second        mixture;    -   c) cooling the second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form I, II,        III, IV, or V of Formula (I), respectively,        wherein the solvent is ethanol or tetrahydrofuran; and the        anti-solvent is ethyl acetate, methyl tert-butyl ether, heptane,        or toluene.

In some embodiments, the present disclosure provides a process forpreparing crystalline Form I of the compound of Formula (I), the processincluding:

-   -   a) forming a first mixture including a compound of Formula (I)        and ethanol at a temperature of at least 20° C.;    -   b) adding an anti-solvent to the first mixture to form a second        mixture;    -   c) cooling the second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form I of        Formula (I),        wherein the anti-solvent is ethyl acetate or toluene.

In some embodiments, the present disclosure provides a process forpreparing crystalline Form I of the compound of Formula (I), the processincluding:

-   -   a) forming a first mixture including a compound of Formula (I)        and ethanol at a temperature of at least 20° C.;    -   b) adding ethyl acetate to the first mixture to form a second        mixture;    -   c) cooling the second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form I of        Formula (I).

In some embodiments, the present disclosure provides a process forpreparing crystalline Form I of the compound of Formula (I), the processincluding:

-   -   a) forming a first mixture including a compound of Formula (I)        and ethanol at a temperature of at least 20° C.;    -   b) adding toluene to the first mixture to form a second mixture;    -   c) cooling the second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form I of        Formula (I).

In some embodiments, the present disclosure provides a process forpreparing crystalline Form II, V, or a mixture of Forms II and V of thecompound of Formula (I), the process including:

-   -   a) forming a first mixture including a compound of Formula (I)        and ethanol at a temperature of at least 20° C.;    -   b) adding heptane to the first mixture to form a second mixture;    -   c) cooling the second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form II of        Formula (I).

In some embodiments, the present disclosure provides a process forpreparing crystalline Form III of the compound of Formula (I), theprocess including:

-   -   a) forming a first mixture including a compound of Formula (I)        and ethanol at a temperature of at least 20° C.;    -   b) adding methyl tert-butyl ether to the first mixture to form a        second mixture;    -   c) cooling the second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form III of        Formula (I).

In some embodiments, the present disclosure provides a process forpreparing crystalline Form IV of the compound of Formula (I), theprocess including:

-   -   a) forming a first mixture including a compound of Formula (I)        and tetrahydrofuran at a temperature of at least 20° C.;    -   b) adding ethyl acetate to the first mixture to form a second        mixture;    -   c) cooling the second mixture and stirring to form a        precipitate;    -   d) isolating the precipitate; and    -   e) drying the precipitate to provide the crystalline Form IV of        Formula (I).

In some embodiments, the first mixture comprises a crude compound ofFormula (I). In other embodiments, the first mixture comprises acrystalline form of a compound of Formula (I), e.g., a crystalline formof a compound of Formula (I) according to this disclosure, or acrystalline form described in WO 2020/123772 (i.e., Form A or Form B).In some embodiments, the first mixture comprises crystalline Form A, B,I, II, III, IV, V or VI. In some embodiments, the first mixturecomprises crystalline Form A or B. In some embodiments, the firstmixture comprises crystalline Form A. In some embodiments, the firstmixture comprises crystalline Form B.

In some embodiments, the first and/or second mixture are a solutionincluding the compound of Formula (I). In some embodiments, the firstand/or second mixture are a solution including the compound of Formula(I) and ethanol. In some embodiments, the first and/or second mixtureare a hazy solution including the compound of Formula (I) and ethanol.In some embodiments, the first and/or second mixture are a hazy solutionincluding the compound of Formula (I) and tetrahydrofuran.

Slow evaporation of a saturated solution of material in an appropriatesolvent or mixture is also effective in obtaining crystalline material.In general, the sample has lower crystallinity as measured by XRPD.Suitable solvents include but are not limited to acetone,tetrahydrofuran, ethanol, methanol, acetonitrile, and water.

Solvent/Antisolvent Ratio

In the case of a binary solvent mixture, the formation of thecrystalline form can be sensitive to the ratio of solvent toprecipitating solvent. For example, if the material is dissolved inethanol and ethyl acetate is added as a precipitating solvent, the finalethanol to ethyl acetate ratio can vary from 4:1 to 1:4, e.g. 4:1, 3:1,2:1, 1:1, 1:2, 1:3 or 1:4. In some embodiments, the final ethanol toethyl acetate ratio is 2:1. Without wishing to be bound by theory,solvent systems characterized by a greater ethanol content than ethylacetate content tend to favor the formation of crystalline Form II. Bycontrast solvent systems characterized by a lower ethanol content thanethyl acetate content tend to favor the formation of crystalline Form I.In one embodiment, the ethanol content is greater than the ethyl acetatecontent. In another embodiment the ethanol content is lower than theethyl acetate content. In some embodiments, the ethanol to ethyl acetateratio is 4:1, 3:1, 2:1 or 1:1. In alternative embodiments the ethanol toethyl acetate content is 1:1, 1:2, 1:3 or 1:4.

In some embodiments, the ethanol to ethyl acetate ratio is from 1:1 to1:4. In some embodiments, the ethanol to ethyl acetate ratio is from 1:1to 1:2. In some embodiments, the ethanol to ethyl acetate ratio is about5:8. In some embodiments, the ethanol to toluene ratio is from 1:1 to1:4. In some embodiments, the ethanol to toluene ratio is about 1:2. Insome embodiments, the ethanol to methyl tert-butyl ether ratio is from1:1 to 1:4. In some embodiments, the ethanol to methyl tert-butyl etherratio is about 1:2. In some embodiments, the ethanol to methyltert-butyl ether ratio is about 5:14. In some embodiments, thetetrahydrofuran to ethyl acetate ratio is from 1.5:1 to 1:2. In someembodiments, the tetrahydrofuran to ethyl acetate ratio is about 5:4. Insome embodiments, the ethanol to heptane ratio is from 1:1 to 1:4.

If slow evaporation is used to obtain crystalline material, a mixture ofsolvents may be used. In some embodiments the ratio of solvents can varyfrom 4:1 to 1:1. In another embodiment, slow evaporation of one solventcan be used to obtain crystalline material.

Solvent/Compound Ratio

The ratio or concentration of compound relative to solvent can bevariable depending on the solvent or solvent mixture used. Typicalconcentrations can range from 250 mg/mL to 10 mg/mL with the limitingfactor at the higher end being the solubility of the material or theease of recovery the material once crystallization has occurred. Forexample, approximately 200 mg of amorphous material can be dissolved in1 mL of ethanol with subsequent addition of an anti-solvent (e.g., ethylacetate, heptane, or toluene) added to afford the crystalline form.

Temperature Control

In general, the maximum heating temperature used in the above methodscan range from 20° C. to the reflux temperature of the solvent. Mosttypical temperatures range from 20° C. to 60° C. Once a solution hasbeen obtained, and, if required, a precipitating solvent added, themixture is cooled to room temperature. The rate of cooling can affectthe size, shape, and quality of the crystals. If the solution issubjected to prolonged heating over 60° C. or contains a reactivesolvent, decomposition can occur.

In some embodiments, step a) is conducted at a temperature of from 20°C. to 100° C. In some embodiments, step a) is conducted at a temperatureof from 40° C. to 80° C. In some embodiments, step a) is conducted at atemperature of from 55° C. to 60° C. In some embodiments, step a) isconducted at a temperature of from 35° C. to 55° C. In some embodiments,step a) is conducted at a temperature from 35° C. to 40° C. In someembodiments, step a) is conducted at a temperature from 20° C. to 25° C.

In some embodiments, when step b) is present, step b) is conducted at atemperature of from 20° C. to 100° C. In some embodiments, when step b)is present, step b) is conducted at a temperature of from 40° C. to 80°C. In some embodiments, when step b) is present, step b) is conducted ata temperature of from 55° C. to 60° C. In some embodiments, when step b)is present, step b) is conducted at a temperature of from 35° C. to 55°C. In some embodiments, when step b) is present, step b) is conducted ata temperature of from 35° C. to 40° C. In some embodiments, when step b)is present, step b) is conducted at a temperature of from 20° C. to 25°C.

In some embodiments, steps a) and b) are each conducted at a temperatureof from about 55° C. to about 60° C. In some embodiments, steps a) andb) are each conducted at a temperature of from about 35° C. to 55° C. Insome embodiments, steps a) and b) are each conducted at a temperature offrom about 35° C. to 40° C. In some embodiments, steps a) and b) areeach conducted at a temperature of from about 20° C. to 25° C.

Rate of Crystallization

Several factors significantly impact the rate of crystallization. Theseinclude, but are not limited to: rate of precipitating solvent addition,rate of mixture cooling, and presence of nucleation sites such as dust,seed crystals, or defects on the glass surface. Variations in theseparameters can affect the size, shape, and quality of the crystals.

In some embodiments, step c) is conducted by c-1) cooling the first orthe second mixture to room temperature for a period of from 30 minutesto 3 hours; and c-2) stirring at room temperature for a period of from12 hours to 72 hours to form the precipitate. In some embodiments, stepc) is conducted by c-1) cooling the first or the second mixture to roomtemperature for a period of from 1 to 2 hours; and c-2) stirring at roomtemperature for a period of about 18 hours to form the precipitate.

In some embodiments, a crystal seed of the compound of Formula (I) isadded during step c).

Isolation of the Crystal Form

Several methods for isolation of the desired crystalline form from thesupernatant can be used including filtration, decantation, and solventevaporation. In general, the crystalline form was obtained by collectingany formed solids by vacuum filtration, followed by air-drying andsubsequent exposure to high vacuum to remove any residual solvent.

In some embodiments, the isolating of step d) is conducted byfiltration.

In some embodiments, the drying of step e) is conducted a temperature offrom about 55° C. to about 60° C. under vacuum. In some embodiments, thedrying of step e) is conducted at ambient temperature, e.g., betweenabout 20° C. and 25° C. under vacuum.

Table 1 summarizes the preparation of crystalline forms of Formula (I).

TABLE 1 Crystallization summary Anti- wt % Solvent solvent Add'n Crudewt % residual (I) (v/w (v/w Addition Temp % Temp residual anti- (g)parts) parts) mode (° C.) yield (° C.) (hrs) solvent solvent XRPD 1.008.0 11.0  Normal^(B) 20 87 40-45 18 0.28 1.34 — THF EtOAc 55-60 48 0.221.05 — 1.00 8.0 Normal^(B) 20 90 40-45 18 1.03 6.96 — THF 8.0 55-60 480.89 6.46 — iPrOAc 1.00 8.0 6.0 Normal^(B) 20 53 40-45 18 0.73 1.43 —THF MTBE 55-60 48 0.46 0.90 — 1.00 8.0 11.0  In- 20 87 55-60 48 0.953.15 Amor- THF EtOAc verse^(C) + phous seeds 1.00 8.0 8.0 In- 20 8955-60 48 0.98 6.10 THF iPrOAc verse^(C) + seeds 1.00 8.0 8.0 In- 20Semi- — — — — — THF MTBE verse^(C) + solid/Gel seeds 1.00 5.0 4.0Normal^(B) ~60  58^(A) 55-60 72 0.06 0.08 Form THF EtOAc IV 1.00 6.0 4.0Normal^(B) ~60 43 55-60 72 1.82 2.14 — THF MTBE 1.00 3.0 5.0 Normal^(D)~60 56 55-60 18 0.06 0.91 Form THF IPA A^(F) 1.00 4.0 5.0 Normal^(D) ~60Emulsion — — — — — THF MEK 1.00 7.0 — — ~60 37 55-60 18 0.68 — Form IIEtOH 1.00 13.0 4.0 All-in^(E) ~60 16 55-60 18 0.12 0.47 THF EtOAc 1.005.0 4.0 Normal^(B) ~60 83 55-60 18 0.09 0.25 THF EtOAc 0.50 5.0 4.0Normal^(B) ~60 Solution — — — — — THF MeTHF + 8 MeTHF 0.50 6.0 10.0Normal^(D) ~60 Solution — — — — — THF IPA + 8 IPA 0.50 5.0 10.0 Normal^(B) ~60 68 55-60 18 ~0 0.40 Form EtOH MTBE III 1.00 5.0 3.0Normal^(B) ~60 75 55-60 72 0.32 1.90 THF Toluene 1.00 5.0 10.0 Normal^(B) ~60 82 55-60 72 0.04 ~0 Form I EtOH Toluene 1.00 5.0 8.0Normal^(B) ~60 82 55-60 72 0.04 0.04 Form I EtOH EtOAc 3.00 5.0 4.0 Nor-55-60 93 55-60 18 0.05 0.38 Form THF EtOAc mal^(B) + IV seeds 3.00 5.014.0  Nor- 55-60 82 55-60 18 0.14 1.56 Form EtOH MTBE mal^(B) + IIIseeds 3.00 5.0 8.0 Nor- 55-60 83 55-60 18 ~0 0.02 Form EtOH EtOAcmal^(B) + I seeds 3.00 5.0 5.0 Normal^(B) 55-60 94 55-60 72 0.16 0.72THF EtOAc 3.00 5.0 10.0  Normal^(B) 55-60 85 55-60 72 0.03 0.15 EtOHEtOAc 1.00 5.0 3.0 Normal^(B) 45-60 94 55-60 20 6.97 0.38 THF DCM reflux1.00 5.0 3.0 Normal^(B) 45-60 32 55-60 20 0.06 ~0 EtOH DCM reflux 1.005.00 10.0  Normal^(B) 55-60 77 55-60 18 0.51 1.16 Form EtOH EtOAc 21.5 hI HOLD 1.00 5.00 10.0  Normal^(B) 55-60 59 55-60 20 0.05 0.39 EtOH,EtOAc 0.25 H₂O 1.00 5.00 10.0  Normal^(B) 55-60 75 55-60 18 0.73 0.93Form EtOH, EtOAc I 0.25 THF, 0.25 MeTHF 1.00 5.00 10.0  Normal^(B) 55-6080 55-60 20 0.49 1.01 EtOH, EtOAc 0.10 H₂O 1.00 5.00 10.0  Normal^(B)55-60 83 55-60 20 0.44 1.00 EtOH, EtOAc 0.10 THF, 0.10 MeTHF 1.00 5.0010.0  Normal^(B) 55-60 84 55-60 20 0.47 1.02 EtOH, EtOAc 0.25 MeTHF^(A)The isolation was not optimized for yield. Some product was leftbehind on flask wall during the filtration. ^(B)Normal addition:addition of the anti-solvent into the compound of Formula (I)/solventbatch. ^(C)Inverse addition: addition of the compound of Formula(I)/solvent into the anti-solvent batch. ^(D)Normal addition: additionof the solvent into the compound of Formula (I)/anti-solvent batch.^(E)All-in: The compound of Formula (I)/solvent/anti-solvent all addedat the start. Additional solvent added at elevated temp to try tosolubilize the compound of Formula (I). ^(F)Form A of the compound ofFormula (I) is disclosed in PCT/US2019/065916.

V. Compositions

The crystalline form of a compound of Formula (I) may be in the form of,or used to prepare compositions (e.g., further processed with one ormore excipients) suitable for administration to a subject. In general,such compositions are “pharmaceutical compositions” including a compoundof Formula (I) and one or more pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients. In certainembodiments, the compound of Formula (I) is present in a therapeuticallyacceptable amount. The pharmaceutical compositions may be used in themethods of the present disclosure; thus, for example, the pharmaceuticalcompositions can be administered ex vivo or in vivo to a subject inorder to practice the therapeutic and prophylactic methods and usesdescribed herein.

The pharmaceutical compositions of the present disclosure can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions may be used in combinationwith other therapeutically active agents or compounds as describedherein in order to treat or prevent the diseases, disorders andconditions as contemplated by the present disclosure.

The pharmaceutical compositions containing the active ingredient (e.g.,a compound of Formula (I)) may be in a form suitable for oral use, forexample, as tablets, capsules, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups, solutions, microbeads or elixirs. Pharmaceuticalcompositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions, and such compositions may contain one or more agents suchas, for example, sweetening agents, flavoring agents, coloring agentsand preserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets, capsules and the like contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be, for example, diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc.

The tablets, capsules and the like suitable for oral administration maybe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods for the preparation of the above-mentionedformulations will be apparent to those skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents may be naturally occurring gums, for example, gum acacia or gumtragacanth; naturally occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of an CD73 inhibitor contemplated by the presentdisclosure (i.e., a compound of Formula (I)), and one or morepharmaceutically and physiologically acceptable formulation agents.Suitable pharmaceutically acceptable or physiologically acceptablediluents, carriers or excipients include, but are not limited to,antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives(e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl,p-hydroxybenzoate), emulsifying agents, suspending agents, dispersingagents, solvents, fillers, bulking agents, detergents, buffers,vehicles, diluents, and/or adjuvants. For example, a suitable vehiclemay be physiological saline solution or citrate buffered saline,possibly supplemented with other materials common in pharmaceuticalcompositions for parenteral administration. Neutral buffered saline orsaline mixed with serum albumin are further exemplary vehicles. Typicalbuffers that may be used in the compositions according to thisdisclosure include, but are not limited to, pharmaceutically acceptableweak acids, weak bases, or mixtures thereof. As an example, the buffercomponents can be water soluble materials such as phosphoric acid,tartaric acids, lactic acid, succinic acid, citric acid, acetic acid,ascorbic acid, aspartic acid, glutamic acid, and salts thereof.Acceptable buffering agents include, for example, a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including liposomes, hydrogels, prodrugsand microencapsulated delivery systems. For example, a time delaymaterial such as glyceryl monostearate or glyceryl stearate alone, or incombination with a wax, may be employed. A variety of drug deliveryapparatus may be used to deliver a crystalline form of a compound ofFormula (I), including implants (e.g., implantable pumps) and cathetersystems, slow injection pumps and devices, all of which are well knownto the skilled artisan.

Depot injections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the crystalline form ofa compound of Formula (I) disclosed herein over a defined period oftime. Depot injections are usually either solid- or oil-based andgenerally comprise at least one of the formulation components set forthherein. One of ordinary skill in the art is familiar with possibleformulations and uses of depot injections.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Acceptable diluents,solvents and dispersion media that may be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. Moreover, fatty acids such as oleic acid, find use inthe preparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

The present disclosure contemplates the administration of a crystallineform of a compound of Formula (I) in the form of suppositories forrectal administration. The suppositories can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Such materials include, but arenot limited to, cocoa butter and polyethylene glycols.

The crystalline forms of the compound of Formula (I) contemplated by thepresent disclosure may be in the form of any other suitablepharmaceutical composition (e.g., sprays for nasal or inhalation use)currently known or developed in the future.

VI. Methods of Use

The present disclosure contemplates the use of a crystalline form of acompound of Formula (I) described herein in the treatment or preventionof a broad range of diseases, disorders and/or conditions, and/or thesymptoms thereof. While particular uses are described in detailhereafter, it is to be understood that the present disclosure is not solimited. Furthermore, although general categories of particulardiseases, disorders and conditions are set forth hereafter, some of thediseases, disorders and conditions may be a member of more than onecategory, and others may not be a member of any of the disclosedcategories.

The use of a crystalline form of a compound of Formula (I) in themethods of treatment described herein encompasses the direct use (e.g.,administration) of a crystalline form according to this disclosure to asubject, as well as the use of a crystalline form in the preparation ofa medicament for the treatment of the indications described herein. Insome embodiments, the crystalline form of the compound described hereinis preserved in the final dosage form administered to a subject. Inother embodiments, the crystalline form may undergo a physical change,for example, to an amorphous form, a different crystalline form, or asolubilized form, prior to administration to a subject.

Oncology-related Disorders. In accordance with the present disclosure, acompound of Formula (I) (e.g., a crystalline form described herein) canbe used to treat or prevent a proliferative condition or disorder,including a cancer, for example, cancer of the uterus, cervix, breast,prostate, testes, gastrointestinal tract (e.g., esophagus, oropharynx,stomach, small or large intestines, colon, or rectum), kidney, renalcell, bladder, bone, bone marrow, skin, head or neck, liver, gallbladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid,brain (e.g., gliomas), ganglia, central nervous system (CNS) andperipheral nervous system (PNS), and cancers of the hematopoietic systemand the immune system (e.g., spleen or thymus). The present disclosurealso provides methods of treating or preventing other cancer-relateddiseases, disorders or conditions, including, for example, immunogenictumors, non-immunogenic tumors, dormant tumors, virus-induced cancers(e.g., epithelial cell cancers, endothelial cell cancers, squamous cellcarcinomas and papillomavirus), adenocarcinomas such as pancreaticadenocarcinoma, lymphomas, carcinomas, melanomas, leukemias, myelomas,sarcomas, teratocarcinomas, chemically-induced cancers, metastasis, andangiogenesis. The disclosure contemplates reducing tolerance to a tumorcell or cancer cell antigen, e.g., by modulating activity of aregulatory T-cell and/or a CD8+ T-cell (see, e.g., Ramirez-Montagut, etal. (2003) Oncogene 22:3180-87; and Sawaya, et al. (2003) New Engl. J.Med. 349:1501-09). In particular embodiments, the tumor or cancer iscolon cancer, ovarian cancer, breast cancer, melanoma, lung cancer,glioblastoma, or leukemia. The use of the term(s) cancer-relateddiseases, disorders and conditions is meant to refer broadly toconditions that are associated, directly or indirectly, with cancer, andincludes, e.g., angiogenesis and precancerous conditions such asdysplasia.

In certain embodiments, a cancer is metastatic or at risk of becomingmetastatic, or may occur in a diffuse tissue, including cancers of theblood or bone marrow (e.g., leukemia). In some further embodiments, thecrystalline form of a compound of Formula (I) can be used to overcomeT-cell tolerance.

In one embodiment, the cancer is a gastrointestinal malignancy such aspancreatic cancer. In one embodiment, the cancer is metastaticpancreatic adenocarcinoma. In one embodiment, a patient is treated forpancreatic cancer using the compound of Formula (I) and an anti-PD-1antibody. In another embodiment, a patient is treated for first linemetastatic pancreatic cancer using a compound of Formula (I) and ananti-PD-1 antibody and standard of care agents for pancreatic cancersuch as those described herein. Patients may be treatment experienced ortreatment naïve.

In some embodiments, the present disclosure provides methods fortreating a proliferative condition, cancer, tumor, or precancerouscondition with a compound of Formula (I) (e.g., a crystalline formdescribed herein) and at least one additional therapeutic or diagnosticagent, examples of which are set forth elsewhere herein.

In some embodiments, the methods described herein may be indicated asfirst line, second line or third line treatments.

Immune-related Disorders and Disorders with an Inflammatory Component.As used herein, terms such as “immune disease”, “immune condition”,“immune disorder”, “inflammatory disease”, “inflammatory condition”,“inflammatory disorder” and the like are meant to broadly encompass anyimmune-related condition (e.g., an autoimmune disease) or a disorderwith an inflammatory component that can be treated by the compound ofFormula (I) (e.g., a crystalline form described herein) such that sometherapeutic benefit is obtained. Such conditions frequently areinextricably intertwined with other diseases, disorders and conditions.By way of example, an “immune condition” may refer to proliferativeconditions, such as cancer, tumors, and angiogenesis; includinginfections (acute and chronic), tumors, and cancers that resisteradication by the immune system.

The compound of Formula (I) (e.g., a crystalline form described herein)can be used to increase or enhance an immune response; to improveimmunization, including increasing vaccine efficacy; and to increaseinflammation. Immune deficiencies associated with immune deficiencydiseases, immunosuppressive medical treatment, acute and/or chronicinfection, and aging can be treated using the compounds disclosedherein. The compound of Formula (I) (e.g., a crystalline form describedherein) can also be used to stimulate the immune system of patientssuffering from iatrogenically-induced immune suppression, includingthose who have undergone bone marrow transplants, chemotherapy, orradiotherapy.

In particular embodiments of the present disclosure, the compound ofFormula (I) (e.g., a crystalline form described herein) is used toincrease or enhance an immune response to an antigen by providingadjuvant activity. In a particular embodiment, at least one antigen orvaccine is administered to a subject in combination with a compound ofFormula (I) (e.g., a crystalline form described herein) to prolong animmune response to the antigen or vaccine. Therapeutic compositions arealso provided which include at least one antigenic agent or vaccinecomponent, including, but not limited to, viruses, bacteria, and fungi,or portions thereof, proteins, peptides, tumor-specific antigens, andnucleic acid vaccines, in combination with a compound of Formula (I)(e.g., a crystalline form described herein).

Microbial-related Disorders. By inhibiting the immunosuppressive andanti-inflammatory activity of CD73, the present disclosure contemplatesthe use of a compound of Formula (I) (e.g., a crystalline form describedherein) in the treatment and/or prevention of any viral, bacterial,fungal, parasitic or other infective disease, disorder or condition forwhich treatment with an CD73 inhibitor may be beneficial. Examples ofsuch diseases and disorders include HIV and AIDS, staphylococcal andstreptococcal infections (e.g., Staphylococcus aureus and streptococcussanguinis, respectively), Leishmania, Toxoplasma, Trichomonas, Giardia,Candida albicans, Bacillus anthracis, and Pseudomonas aeruginosa.Compounds of the disclosure can be used to treat sepsis, decrease orinhibit bacterial growth, and reduce or inhibit inflammatory cytokines.

CNS-related and Neurological Disorders. Inhibition of CD73 may also bean important treatment strategy for patients with neurological,neuropsychiatric, neurodegenerative or other diseases, disorders andconditions having some association with the central nervous system,including disorders associated with impairment of cognitive function andmotor function. Examples include Parkinson's disease, extra pyramidalsyndrome (EPS), dystonia, akathisia, tardive dyskinesia, restless legsyndrome (RLS), epilepsy, periodic limb movement in sleep (PLMS),attention deficit disorders, depression, anxiety, dementia, Alzheimer'sdisease, Huntington's disease, multiple sclerosis, cerebral ischemia,hemorrhagic stroke, subarachnoid hemorrhage, and traumatic brain injury.

Other Disorders. Embodiments of the present disclosure contemplate theadministration of a compound of Formula (I) (e.g., a crystalline formdescribed herein) to a subject for the treatment or prevention of anyother disorder that may benefit from at least some level of CD73inhibition. Such diseases, disorders and conditions include, forexample, cardiovascular (e.g., cardiac ischemia), gastrointestinal(e.g., Crohn's disease), metabolic (e.g., diabetes), hepatic (e.g.,hepatic fibrosis, NASH, and NAFLD), pulmonary (e.g., COPD and asthma),ophthalmologic (e.g., diabetic retinopathy), and renal (e.g., renalfailure) disorders.

In some embodiments, a compound of Formula (I) (e.g., a crystalline formdescribed herein) may be used to inhibit statin-induced adenosineproduction, or reduce or decrease increases in blood glucose caused by astatin in a subject taking a statin (e.g., lovastatin and pravastatin).

Selection of Patients.

In some instances, the methods according to this disclosure may beindicated in certain patients, for example based on CD73 as a biomarker,high microsatellite instability, or high tumor mutational burden. Insome instances, the subject is identified as having an oncogene drivenor oncogene addicted cancer that has a mutation in at least one geneassociated with CD73. Methods of testing determining CD73 levels and thepresence of CD73 associated oncogenes are disclosed in WO 2020/185859and WO 2020/205527.

Routes of Administration

The present disclosure contemplates the administration of a crystallineform of a compound of Formula (I), and compositions thereof, in anyappropriate manner. In one or more embodiments, the crystalline form ofa compound of Formula (I) is useful in the manufacture of a medicamentsuitable for administration to a subject. In some embodiments, thecrystalline form of the compound of Formula (I) is preserved in themedicament administered to a subject. In other embodiments, thecrystalline form of the compound of Formula (I) undergoes a physicalchange, e.g., to an amorphous form, or a different crystalline form,during preparation of the medicament. Suitable routes of administrationinclude oral, parenteral (e.g., intramuscular, intravenous, subcutaneous(e.g., injection or implant), intraperitoneal, intracisternal,intraarticular, intraperitoneal, intracerebral (intraparenchymal) andintracerebroventricular), nasal, vaginal, sublingual, intraocular,rectal, topical (e.g., transdermal), buccal and inhalation. Depotinjections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the crystalline form ofa compound of Formula (I) disclosed herein over a defined period oftime.

Particular embodiments of the present disclosure contemplate oraladministration. Other embodiments of the present disclosure contemplateparenteral administration.

5′-Nucleotidase, Ecto and Inhibition Thereof

Human CD73 (also referred to as 5′-nucleotidase, ecto; NTSE; or 5NT) isa 574 amino acid residue protein (Accession No. AAH6593). EukaryoticCD73 functions as a noncovalent homodimer with two structural domains,wherein the N- and C-terminal domains are connected by a hinge regionthat enables the enzyme to undergo large domain movements and switchbetween open and closed conformations (Knapp, K. et al. (2012) Structure20:2161-73).

CD73 inhibitors can modulate purinergic signaling, a type ofextracellular signaling mediated by purine nucleotides and nucleosidessuch as ATP and adenosine. Purinergic signaling involves the activationof purinergic receptors in the cell and/or in nearby cells, resulting inthe regulation of cellular functions. The enzymatic activity of CD73plays a strategic role in calibrating the duration, magnitude, andchemical nature of purinergic signals delivered to various cells (e.g.,immune cells). Alteration of these enzymatic activities can change thecourse or dictate the outcome of several pathophysiological events,including cancer, autoimmune and inflammatory diseases, infections,atherosclerosis, and ischemia-reperfusion injury, suggesting that theseecto-enzymes represent novel therapeutic targets for managing a varietyof disorders.

Studies using tissues that overexpress CD73 and using CD73 knock-outmice have provided evidence that CD73 inhibitors have potential utilityfor melanomas, lung cancer, prostate cancer, and breast cancer (see,e.g., Sadej R. (2006) Melanoma Res 16:213-22). Because higher expressionlevels of CD73 are associated with tumor neovascularization,invasiveness, resistance to chemotherapy, and metastasis, CD73inhibitors can be used to control tumor progression and metastasis.Other potential utilities are discussed elsewhere herein.

Although the compound of Formula (I) is believed to exert its activityby inhibition of CD73, a precise understanding of the compound'sunderlying mechanism of action is not required to practice thedisclosure. For example, the compound can also exert its activity, atleast in part, through modulation (e.g., inhibition) of other componentsof the purinergic signaling pathway (e.g., CD39). The purinergicsignaling system consists of transporters, enzymes and receptorsresponsible for the synthesis, release, action, and extracellularinactivation of (primarily) ATP and its extracellular breakdown productadenosine (Sperlagh, B. et al. (December 2012) NeuropsychopharmacologiaHungarica 14(4):231-38). There are several potential opportunities formodulation of the signaling process. However, some of theseopportunities are more tractable than others.

VII. Combination Therapy

The present disclosure contemplates the use of a compound of Formula (I)(e.g., a crystalline form described herein) in combination with one ormore active therapeutic agents. The additional active therapeutic agentscan be small chemical molecules; macromolecules such as proteins,antibodies, peptibodies, peptides, DNA, RNA or fragments of suchmacromolecules; or cellular or gene therapies. In such combinationtherapy, the various active agents frequently have different,complementary mechanisms of action. Such combination therapy may beadvantageous by allowing a dose reduction of one or more of the agents,thereby reducing or eliminating the adverse effects associated with oneor more of the agents. The compound of Formula (I) of the presentdisclosure may also be useful in overcoming adenosine-dependentimmunosuppression, leading to enhanced therapeutic efficacy of otheragents. Furthermore, such combination therapy may have a synergistictherapeutic or prophylactic effect on the underlying disease, disorder,or condition.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as may be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”).

In certain embodiments, a compound of Formula (I) (e.g., a crystallineform described herein) is administered or applied sequentially, e.g.,where one agent is administered prior to one or more other agents. Inother embodiments, the agents are administered simultaneously, e.g.,where two or more agents are administered at or about the same time; thetwo or more agents may be present in two or more separate formulationsor combined into a single formulation (i.e., a co-formulation).Regardless of whether the two or more agents are administeredsequentially or simultaneously, they are considered to be administeredin combination for purposes of the present disclosure.

The compound of Formula (I) (e.g., a crystalline form described herein)may be used in combination with at least one other (active) agent in anymanner appropriate under the circumstances. In one embodiment, treatmentwith the at least one active agent and the crystalline form of thecompound of Formula (I) is maintained over a period of time. In anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment withthe crystalline form of the compound of Formula (I) is maintained at aconstant dosing regimen. In a further embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with the crystalline form of thecompound of Formula (I) is reduced (e.g., lower dose, less frequentdosing or shorter treatment regimen). In yet another embodiment,treatment with the at least one active agent is reduced or discontinued(e.g., when the subject is stable), and treatment with the crystallineform of the compound of Formula (I) is increased (e.g., higher dose,more frequent dosing or longer treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is maintainedand treatment with the crystalline form of the compound of Formula (I)is reduced or discontinued (e.g., lower dose, less frequent dosing orshorter treatment regimen). In yet another embodiment, treatment withthe at least one active agent and treatment with the crystalline form ofthe compound of Formula (I) is reduced or discontinued (e.g., lowerdose, less frequent dosing or shorter treatment regimen).

Oncology-related Disorders. The present disclosure provides methods fortreating and/or preventing a proliferative condition, cancer, tumor, orprecancerous disease, disorder or condition with a compound of Formula(I) (e.g., a crystalline form according to this disclosure) and at leastone additional therapeutic or diagnostic agent.

In some embodiments, one or more of the additional therapeutic agents isan immunomodulatory agent. Suitable immunomodulatory agents that may beused in the present disclosure target CD40L, B7, and B7RP1; activatingmonoclonal antibodies (mAbs) to stimulatory receptors, such as,anti-CD40, anti-CD38, anti-ICOS, and 4-IBB ligand; dendritic cellantigen loading (in vitro or in vivo); anti-cancer vaccines such asdendritic cell cancer vaccines; cytokines/chemokines, such as, IL1, IL2,IL12, IL18, ELC/CCL19, SLC/CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC,IFNa/b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacteriallipopolysaccharides (LPS); indoleamine 2,3-dioxygenase 1 (IDO1)inhibitors and immune-stimulatory oligonucleotides.

In certain embodiments, the present disclosure provides methods fortumor suppression of tumor growth including administration of a compoundof Formula (I) (e.g., as a crystalline form described herein) incombination with a signal transduction inhibitor (STI) to achieveadditive or synergistic suppression of tumor growth. As used herein, theterm “signal transduction inhibitor” refers to an agent that selectivelyinhibits one or more steps in a signaling pathway. Signal transductioninhibitors (STIs) contemplated by the present disclosure include: (i)BCR-ABL kinase inhibitors (e.g., GLEEVEC®); (ii) epidermal growth factorreceptor tyrosine kinase inhibitors (EGFR TKIs), including smallmolecule inhibitors (e.g., gefitinib, erlotinib, afatinib, andosimertinib), and anti-EGFR antibodies; (iii) inhibitors of the humanepidermal growth factor (HER) family of transmembrane tyrosine kinases,e.g., HER-2/neu receptor inhibitors (e.g., HERCEPTIN®), and HER-3receptor inhibitors; (iv) vascular endothelial growth factor receptor(VEGFR) inhibitors including small molecule inhibitors (e.g., axitinib,sunitinib and sorafenib), and anti-VEGF antibodies (e.g., bevacizumab);(v) inhibitors of AKT family kinases or the AKT pathway (e.g.,rapamycin); (vi) inhibitors of serine/threonine-protein kinase B-Raf(BRAF), such as, for example, vemurafenib, dabrafenib and encorafenib;(vii) inhibitors of rearranged during transfection (RET), including, forexample, selpercatinib and pralsetinib; (viii) tyrosine-protein kinaseMet (MET) inhibitors (e.g., tepotinib, tivantinib, cabozantinib andcrizotinib); (ix) anaplastic lymphoma kinase (ALK) inhibitors (e.g.,ensartinib, ceritinib, lorlatinib, crizotinib, and brigatinib); (x)inhibitors of the RAS signaling pathway (e.g., inhibitors of KRAS, HRAS,RAF, MEK, ERK) as described elsewhere herein; (xi) FLT-3 inhibitors(e.g., gilteritinib); (xii) inhibitors of Trop-2, such as, for example,the antibody drug conjugate sacituzumab govitecan-hziy; (xiii)inhibitors of the JAK/STAT pathway, e.g., JAK inhibitors includingtofacitinib and ruxolitinib, or STAT inhibitors such as napabucasin;(xiv) inhibitors of NF-κB; (xv) cell cycle kinase inhibitors (e.g.,flavopiridol); (xvi) phosphatidyl inositol kinase (PI3K) inhibitors; and(xix) protein kinase B (AKT) inhibitors (e.g., capivasertib,miransertib). Agents involved in immunomodulation can also be used incombination with the crystal forms described herein for the suppressionof tumor growth in cancer patients. In one or more embodiments, theadditional therapeutic agent comprises an inhibitor of EGFR, VEGFR,HER-2, HER-3, BRAF, RET, MET, ALK, RAS (e.g., KRAS, MEK, ERK), FLT-3,JAK, STAT, NF-κB, PI3K, AKT, or any combinations thereof.

In other embodiments, the present disclosure provides methods fortreating cancer in a subject, including administering to the subject atherapeutically effective amount of a compound of Formula (I) (e.g., asa crystalline form described herein) and at least one chemotherapeuticagent. Examples of chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphaoramide andtrimethylolomelamime; nitrogen mustards such as chiorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate and 5-fluorouracil (5-FU) with or without leucovorin; folicacid analogs such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine, 5-FU; androgens such ascalusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as folinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elformithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa;taxoids, e.g., paclitaxel, nab-paclitaxel, and docetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum andplatinum coordination complexes such as cisplatin and carboplatin;vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; anthracyclines; and pharmaceutically acceptable salts,acids or derivatives of any of the above.

Chemotherapeutic agents also include anti-hormonal agents that act toregulate or inhibit hormonal action on tumors such as anti-estrogens,including for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone,and toremifene; and antiandrogens such as abiraterone, apalutamide,darolutamide, flutamide, nilutamide, bicalutamide, leuprolide,enzalutamide, and goserelin; and pharmaceutically acceptable salts,acids or derivatives of any of the above. In certain embodiments,combination therapy includes administration of a hormone or relatedhormonal agent.

In some embodiments, the additional chemotherapeutic agent is selectedfrom nab-paclitaxel, gemcitabine and combinations thereof. In someembodiments, the additional chemotherapeutic agent is selected fromenzalutamide, docetaxel and combinations thereof.

In some embodiments drawn to methods of treating cancer, theadministration of a therapeutically effective amount of a compound ofFormula (I) (e.g., as a crystalline form described herein) incombination with at least one chemotherapeutic agent results in a cancersurvival rate greater than the cancer survival rate observed byadministering either agent alone. In further embodiments drawn tomethods of treating cancer, the administration of a therapeuticallyeffective amount of a compound of Formula (I) in combination with atleast one chemotherapeutic agent results in a reduction of tumor size ora slowing of tumor growth greater than reduction of the tumor size ortumor growth observed by administration of either agent alone.

Combinations of a compound of Formula (I) (e.g., a crystalline formdescribed herein) with a poly (ADP-ribose) polymerase (PARP) inhibitoris also contemplated. Exemplary PARP inhibitors contemplated by thisdisclosure include olaparib, niraparib and rucaparib.

In one or more embodiments, combinations of a compound of Formula (I)(e.g., a crystalline form described herein) with inhibitors of the Bcl-2family of proteins, such as, for example inhibitors of BCL-2 (e.g.,venetoclax and navitoclax), and inhibitors of MCL-1 are alsocontemplated.

Combinations of a compound of Formula (I) (e.g., a crystalline formdescribed herein) with inhibitors of the CD47-SIRPα pathway (e.g., theanti-CD47 antibody, magrolimab) are also contemplated.

In one or more embodiments, combinations of a compound of Formula (I)(e.g., a crystalline form described herein) with DNA methyltransferase(DNMT) inhibitors or hypomethylating agents is also contemplated.Exemplary DNMT inhibitors include decitabine, zebularine andazacitadine.

In one or more embodiments, combinations of a compound of Formula (I)(e.g., a crystalline form described herein) with a histone deacetylase(HDAC) inhibitor is also contemplated. Exemplary HDAC inhibitors includevorinostat, givinostat, abexinostat, panobinostat, belinostat andtrichostatin A.

In some embodiments, a compound of Formula (I) (e.g., a crystalline formdescribed herein) are combined with a menin-MLL inhibitor.

In some embodiments, combination of a compound of Formula (I) (e.g., acrystalline form described herein) with a isocitrate dehydrogenase (IDH)inhibitor, e.g., IDH-1 or IDH-2, is also contemplated. An exemplaryIDH-1 inhibitor is ivosidenib. An exemplary IDH-2 inhibitor isenasidenib.

Additional treatment modalities that may be used in combination with athe compound of Formula (I) (e.g., a crystalline form described herein)include radiotherapy, surgical resection, a monoclonal antibody againsta tumor antigen, a complex of a monoclonal antibody and toxin, a T-celladjuvant, bone marrow transplant, or antigen presenting cells (e.g.,dendritic cell therapy) including toll-like receptor (TLR) agonistswhich are used to stimulate such antigen presenting cells.

Immune Checkpoint Inhibitors. The present disclosure contemplates theuse of the compound of Formula (I) (e.g., a crystalline form describedherein) in combination with immune checkpoint inhibitors.

The tremendous number of genetic and epigenetic alterations that arecharacteristic of all cancers provides a diverse set of antigens thatthe immune system can use to distinguish tumor cells from their normalcounterparts. In the case of T cells, the ultimate amplitude (e.g.,levels of cytokine production or proliferation) and quality (e.g., thetype of immune response generated, such as the pattern of cytokineproduction) of the response, which is initiated through antigenrecognition by the T-cell receptor (TCR), is regulated by a balancebetween co-stimulatory and inhibitory signals (immune checkpoints).Under normal physiological conditions, immune checkpoints are crucialfor the prevention of autoimmunity (i.e., the maintenance ofself-tolerance) and also for the protection of tissues from damage whenthe immune system is responding to pathogenic infection. The expressionof immune checkpoint proteins can be dysregulated by tumors as animportant immune resistance mechanism.

Examples of immune checkpoints (ligands and receptors), some of whichare selectively upregulated in various types of tumor cells, that arecandidates for blockade include PD-1 (programmed cell death protein 1);PD-L1 (PD-1 ligand); BTLA (B and T lymphocyte attenuator); CTLA-4(cytotoxic T-lymphocyte associated antigen 4); TIGIT (T cellimmunoreceptor with Ig and ITIM domains); TIM-3 (T-cell membrane protein3); LAG-3 (lymphocyte activation gene 3); A2aR (adenosine A2a receptorA2aR); and Killer Inhibitory Receptors, which can be divided into twoclasses based on their structural features: i) killer cellimmunoglobulin-like receptors (KIRs), and ii) C-type lectin receptors(members of the type II transmembrane receptor family). Other lesswell-defined immune checkpoints have been described in the literature,including both receptors (e.g., the 2B4 (also known as CD244) receptor)and ligands (e.g., certain B7 family inhibitory ligands such B7-H3 (alsoknown as CD276) and B7-H4 (also known as B7-S1, B7x and VCTN1)). SeePardoll, (April 2012) Nature Rev. Cancer 12:252-64.

The present disclosure contemplates the use of the compound of Formula(I) (e.g., a crystalline form described herein) in combination withinhibitors of the aforementioned immune-checkpoint receptors andligands, as well as yet-to-be-described immune-checkpoint receptors andligands. Certain modulators of immune checkpoints are currentlyavailable, whereas others are in late-stage development. To illustrate,when it was approved for the treatment of melanoma in 2011, the fullyhumanized CTLA-4 monoclonal antibody ipilimumab (YERVOY®; Bristol-MyersSquibb) became the first immune checkpoint inhibitor to receiveregulatory approval in the US. Fusion proteins comprising CTLA-4 and anantibody (CTLA4-Ig; abatcept (ORENCIA®; Bristol-Myers Squibb)) have beenused for the treatment of rheumatoid arthritis, and other fusionproteins have been shown to be effective in renal transplantationpatients that are sensitized to Epstein Barr Virus. The next class ofimmune checkpoint inhibitors to receive regulatory approval were againstPD-1 and its ligands PD-L1 and PD-L2. Approved anti-PD1 antibodiesinclude nivolumab (OPDIVO; Bristol-Myers Squibb) and pembrolizumab(KEYTRUDA®; Merck) for various cancers, including squamous cellcarcinoma, classical Hodgkin lymphoma and urothelial carcinoma. Approvedanti-PDL1 antibodies include avelumab (BAVENCIO®, EMD Serono & Pfizer),atezolizumab (TECENTRIQ®; Roche/Genentech), and durvalumab (IMFINZI®,AstraZeneca) for certain cancers, including urothelial carcinoma. Whilethere are no approved therapeutics targeting TIGIT or its ligands CD155and CD112, those in development include BMS-986207 (Bristol-MyersSquibb), MTIG7192A/RG6058 (Roche/Genentech), OMP-31M32 (OncoMed), anddomvanalimab (AB154). In some combinations provided herein, the immunecheckpoint inhibitor is selected from ipilmumab, tremelimumab,BMS-986016, IMP-731, IMP-321, cobolimab, MBG453, Sym023, INCAGN2390,LY3321367, BMS, 986258, SHR1702, MEDI-0680, pidilizumab (CT-011),nivolumab, pembrolizumab, avelumab, atezolizumab, budigalimab, BI-75091,camrelizumab, cosibelimab, durvalumab, dostarlimab, cemiplimab,sintilimab, tislelizumab, toripalimab, retifanlimab, sasanlimab,domvanalimab (AB154), and zimberelimab (AB122).

In one aspect of the present disclosure, the compound of Formula (I)(e.g., a crystalline form described herein) is combined with animmuno-oncology agent that is (i) an agonist of a stimulatory (includinga co-stimulatory) receptor or (ii) an antagonist of an inhibitory(including a co-inhibitory) signal on T cells, both of which result inamplifying antigen-specific T cell responses. Certain of the stimulatoryand inhibitory molecules are members of the immunoglobulin super family(IgSF). One important family of membrane-bound ligands that bind toco-stimulatory or co-inhibitory receptors is the B7 family, whichincludes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L),B7-H3, B7-H4, B7-H5 (VISTA), B7-H6, and B7-H7 (HHLA2). Another family ofmembrane bound ligands that bind to co-stimulatory or co-inhibitoryreceptors is the TNF family of molecules that bind to cognate TNFreceptor family members, which includes CD40 and CD40L, OX-40, OX-40L,CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L,TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL,TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT13R, LIGHT,DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1,Lymphotoxin a/TNF13, TNFR2, TNFa, LT13R, Lymphotoxin a 1132, FAS, FASL,RELT, DR6, TROY, NGFR.

In another aspect, the immuno-oncology agent is a cytokine that inhibitsT cell activation (e.g., IL-6, IL-10, TGF-B, VEGF, and otherimmunosuppressive cytokines) or a cytokine that stimulates T cellactivation, for stimulating an immune response.

In one aspect, T cell responses can be stimulated by a combination of anoral formulation comprising a compound of Formula (I) and a chelatingagent and one or more of (i) an antagonist of a protein that inhibits Tcell activation (e.g., immune checkpoint inhibitors) such as CTLA-4,PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69,Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1,TIM-1, and TIM-4, and/or (ii) an agonist of a protein that stimulates Tcell activation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS,ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD2. Otheragents that can be combined with an oral formulation comprising acompound of Formula (I) and a chelating agent for the treatment ofcancer include antagonists of inhibitory receptors on NK cells oragonists of activating receptors on NK cells. For example, compoundsherein can be combined with antagonists of KIR, such as lirilumab.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716,WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

In another aspect, the compound of Formula (I) (e.g., a crystalline formdescribed herein) can be used with one or more of agonistic agents thatligate positive costimulatory receptors, blocking agents that attenuatesignaling through inhibitory receptors, antagonists, and one or moreagents that increase systemically the frequency of anti-tumor T cells,agents that overcome distinct immune suppressive pathways within thetumor microenvironment (e.g., block inhibitory receptor engagement(e.g., PD-L1/PD-1 interactions), deplete or inhibit Tregs (e.g., usingan anti-CD25 monoclonal antibody (e.g., daclizumab) or by ex vivoanti-CD25 bead depletion), or reverse/prevent T cell anergy orexhaustion) and agents that trigger innate immune activation and/orinflammation at tumor sites.

Immune Modulators. The present disclosure contemplates the use of thecompound of Formula (I) (e.g., a crystalline form described herein) incombination with therapeutic agents that modulate the tumormicroenvironment or augment or mediate immune responses. Examples ofthese agents include indoleamine 2,3-dioxygenase 1 (IDO-1) inhibitors,adenosine receptor antagonists and arginase inhibitors. IDO-1 breaksdown tryptophan which impairs the activation of anti-tumor T cells.Similarly, arginase has been shown to be responsible for tumor immuneescape through ARG-1, which depletes arginine from the tumormicroenvironment leading to impaired T cell function such as stoppedproliferation and secretion of cytokines. Exemplary arginase compoundscan be found, for example, in WO 2019/173188 and WO 2020/102646.Adenosine signaling through A_(2A)R and A_(2B)R leads to the impairmentof maturation and/or activation of T cells, NK cells and dendriticcells, which then impairs the activation of the immune system againstcancer cells. In some embodiments, the present disclosure contemplatescombination with the adenosine receptor antagonists described inWO/2018/136700, WO 2018/204661, WO 2018/213377, or WO/2020/023846.

In certain embodiments, the present disclosure contemplates the use of acompound of Formula (I) (e.g., a crystalline form described herein) incombination with other agents that modulate the level of adenosine. Suchtherapeutic agents may act on the other ectonucleotides that catalyzethe conversion of ATP to adenosine, including ectonucleosidetriphosphate diphosphohydrolase 1 (ENTPD1, also known as CD39 or Clusterof Differentiation 39), which hydrolyzes ATP to ADP and ADP to AMP.

In certain embodiments, the present invention contemplates the use acompound of Formula (I) (e.g., a crystalline form described herein) withinhibitors of HIF-2α, which plays an integral role in cellular responseto low oxygen availability. Under hypoxic conditions, thehypoxia-inducible factor (HIF) transcription factors can activate theexpression of genes that regulate metabolism, angiogenesis, cellproliferation and survival, immune evasion, and inflammatory response.HIF-2α overexpression has been associated with poor clinical outcomes inpatients with various cancers; hypoxia is also prevalent in many acuteand chronic inflammatory disorders, such as inflammatory bowel diseaseand rheumatoid arthritis. Exemplary HIF-2a inhibitors includebelzutifan, ARC-HIF2, PT-2385, and those described in PCT/US2020/063000and PCT/US2021/022912.

In certain embodiments, the present disclosure contemplates the use of acompound of Formula (I) (e.g., a crystalline form described herein) incombination with inhibitors of phosphatidylinositol 3-kinases (PI3Ks),particularly the PI3Kγ isoform. PI3Kγ inhibitors can stimulate ananti-cancer immune response through the modulation of myeloid cells,such as by inhibiting suppressive myeloid cells, dampeningimmune-suppressive tumor-infiltrating macrophages or by stimulatingmacrophages and dendritic cells to make cytokines that contribute toeffective T-cell responses leading to decreased cancer development andspread. In one embodiment, the PI3Kγ inhibitor is IPI-549. In anotherembodiment the PI3K inhibitor is chosen from those described inPCT/US2020/035920.

The present disclosure also contemplates the combination of a compoundof Formula (I) (e.g., a crystalline form described herein) with one ormore RAS signaling inhibitors. Oncogenic mutations in the RAS family ofgenes, e.g., HRAS, KRAS, and NRAS, are associated with a variety ofcancers. For example, mutations of G12C, G12D, G12V, G12A, G13D, Q61H,G13C and G12S, among others, in the KRAS family of genes have beenobserved in multiple tumor types. Direct and indirect inhibitionstrategies have been investigated for the inhibition of mutant RASsignaling. Indirect inhibitors target effectors other than RAS in theRAS signaling pathway, and include, but are not limited to, inhibitorsof RAF, MEK, ERK, PI3K, PTEN, SOS (e.g., SOS1), mTORC1, SHP2 (PTPN11),and AKT. Non-limiting examples of indirect inhibitors under developmentinclude RMC-4630, RMC-5845, RMC-6291, RMC-6236, JAB-3068, JAB-3312,TN0155, RLY-1971, BI1701963. Direct inhibitors of RAS mutants have alsobeen explored, and generally target the KRAS-GTP complex or the KRAS-GDPcomplex. Exemplary direct RAS inhibitors under development include, butare not limited to, sotorasib (AMG510), MRTX849, mRNA-5671 and ARS1620.In some embodiments, the one or more RAS signaling inhibitors areselected from the group consisting of RAF inhibitors, MEK inhibitors,ERK inhibitors, PI3K inhibitors, PTEN inhibitors, SOS1 inhibitors,mTORC1 inhibitors, SHP2 inhibitors, and AKT inhibitors. In otherembodiments the one or more RAS signaling inhibitors directly inhibitRAS mutants.

In some embodiments, this disclosure is directed to the combination of acompound of Formula (I) (e.g., a crystalline form described herein) withone or more inhibitors of anexelekto (i.e., AXL). The AXL signalingpathway is associated with tumor growth and metastasis, and is believedto mediate resistance to a variety of cancer therapies. There are avariety of AXL inhibitors under development that also inhibit otherkinases in the TAM family (i.e., TYRO3, MERTK), as well as otherreceptor tyrosine kinases including MET, FLT3, RON and AURORA, amongothers. Exemplary multikinase inhibitors include gilteritinib,merestinib, cabozantinib, BMS777607, and foretinib. AXL specificinhibitors have also been developed, e.g., SGI-7079, TP-0903 (i.e.,dubermatinib), BGB324 (i.e., bemcentinib) and DP3975.

The present disclosure also contemplates the combination of a compoundof Formula (I) (e.g., a crystalline form described herein) with one ormore p21-activated kinase 4 (PAK4) inhibitors. PAK4 overexpression hasbeen shown across a variety of cancer types, notably including thoseresistant to PD-1 therapies. While no PAK4 inhibitors have beenapproved, some are in development, and exhibit dual PAK4/NAMPT inhibitoractivity, e.g., ATG-019 and KPT-9274. In some embodiments, the compoundsaccording to this disclosure are combined with a PAK4 selectiveinhibitor. In some embodiments, the compounds according to thisdisclosure are combined with a PAK4/NAMPT dual inhibitor, e.g., ATG-019or KPT-9274.

Metabolic and Cardiovascular Diseases. The present disclosure providesmethods for treating and/or preventing certain cardiovascular- and/ormetabolic-related diseases, disorders and conditions, as well asdisorders associated therewith, with a compound of Formula (I) (e.g., acrystalline form described herein) and at least one additionaltherapeutic or diagnostic agent.

Examples of therapeutic agents useful in combination therapy for thetreatment of hypercholesterolemia (and atherosclerosis as well) includestatins (e.g., CRESTOR®, LESCOL®, LIPITOR®, MEVACOR®, PRAVACOL®, andZOCOR®), which inhibit the enzymatic synthesis of cholesterol; bile acidresins (e.g., COLESTID, LO-CHOLEST, PREVALITE®, QUESTRAN®, andWELCHOL®), which sequester cholesterol and prevent its absorption;ezetimibe (ZETIA®), which blocks cholesterol absorption; fibric acid(e.g., TRICOR®), which reduces triglycerides and may modestly increaseHDL; niacin (e.g., NIACOR®), which modestly lowers LDL cholesterol andtriglycerides; and/or a combination of the aforementioned (e.g.,VYTORIN® (ezetimibe with simvastatin). Alternative cholesteroltreatments that may be candidates for use in combination with the CD73inhibitors described herein include various supplements and herbs (e.g.,garlic, policosanol, and guggul).

The present disclosure encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Immune-related Disorders and Disorders Having an Inflammatory Component.The present disclosure provides methods for treating and/or preventingimmune-related diseases, disorders and conditions; and diseases,disorders and conditions having an inflammatory component; with acompound of Formula (I) (e.g., a crystalline form described herein) andat least one additional therapeutic or diagnostic agent.

Examples of therapeutic agents useful in combination therapy for immune-and inflammatory-related diseases, disorders or conditions include, butare not limited to, the following: non-steroidal anti-inflammatory drug(NSAID) such as aspirin, ibuprofen, and other propionic acid derivatives(alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen,fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen,naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid,and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin,alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid,fentiazac, fuirofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac,tolmetin, zidometacin, and zomepirac), fenamic acid derivatives(flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid andtolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal andflufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican),salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones(apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone,phenylbutazone). Other combinations include cyclooxygenase-2 (COX-2)inhibitors.

Other active agents for combination include steroids such asprednisolone, prednisone, methylprednisolone, betamethasone,dexamethasone, or hydrocortisone. Such a combination may be especiallyadvantageous since one or more adverse effects of the steroid can bereduced or even eliminated by tapering the steroid dose required.

Additional examples of active agents that may be used in combinationsfor treating, for example, rheumatoid arthritis, include cytokinesuppressive anti-inflammatory drug(s) (CSAIDs); antibodies to, orantagonists of, other human cytokines or growth factors, for example,TNF, LT, IL-10, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, or PDGF.

Particular combinations of active agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade, andinclude TNF antagonists such as chimeric, humanized or human TNFantibodies, REMICADE®, HUMIRA®, anti-TNF antibody fragments (e.g.,CDP870), and soluble p55 or p75 TNF receptors, derivatives thereof,p75TNFRIgG (ENBREL®) or p55TNFR1gG (LENERCEPT), soluble IL-13 receptor(sIL-13), and also TNFa-converting enzyme (TACE) inhibitors; similarly,IL-1 inhibitors (e.g., Interleukin-1-converting enzyme inhibitors) maybe effective. Other combinations include Interleukin 11, anti-P7s andp-selectin glycoprotein ligand (PSGL). Other examples of agents usefulin combination with the crystalline forms described herein includeinterferon-131a (AVONEX®); interferon-131b (BETASERON®); copaxone;hyperbaric oxygen; intravenous immunoglobulin; clabribine; andantibodies to, or antagonists of, other human cytokines or growthfactors (e.g., antibodies to CD40 ligand and CD80).

Microbial Diseases. The present disclosure provides methods for treatingand/or preventing viral, bacterial, fungal and parasitic diseases,disorders and conditions, as well as disorders associated therewith,with a compound of Formula (I) (e.g., a crystalline form describedherein) and at least one additional therapeutic or diagnostic agent(e.g., one or more other antiviral agents and/or one or more agents notassociated with viral therapy).

Such combination therapy includes anti-viral agents targeting variousviral life-cycle stages and having different mechanisms of action,including, but not limiting to, the following: inhibitors of viraluncoating (e.g., amantadine and rimantidine); reverse transcriptaseinhibitors (e.g., acyclovir, zidovudine, and lamivudine); agents thattarget integrase; agents that block attachment of transcription factorsto viral DNA; agents (e.g., antisense molecules) that impact translation(e.g., fomivirsen); agents that modulate translation/ribozyme function;protease inhibitors; viral assembly modulators (e.g., rifampicin);antiretrovirals such as, for example, nucleoside analogue reversetranscriptase inhibitors (e.g., azidothymidine (AZT), ddl, ddC, 3TC,d4T); non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz,nevirapine); nucleotide analogue reverse transcriptase inhibitors; andagents that prevent release of viral particles (e.g., zanamivir andoseltamivir). Treatment and/or prevention of certain viral infections(e.g., HIV) frequently entail a group (“cocktail”) of antiviral agents.

Other antiviral agents contemplated for use in combination with acompound of Formula (I) (e.g., a crystalline form described herein)include, but are not limited to, the following: abacavir, adefovir,amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla,boceprevirertet, cidofovir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, emtricitabine, enfuvirtide, entecavir,famciclovir, fosamprenavir, foscarnet, fosfonet, ganciclovir,ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine,various interferons (e.g., peginterferon alfa-2a), lopinavir, loviride,maraviroc, moroxydine, methisazone, nelfinavir, nexavir, penciclovir,peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin,ritonavir, pyramidine, saquinavir, stavudine, telaprevir, tenofovir,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, and zalcitabine.

The present disclosure contemplates the use of the compound of Formula(I) (e.g., a crystalline form described herein) in combination withantiparasitic agents. Such agents include, but are not limited to,thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide,bithionol, oxamniquine, metrifonate, ivermectin, albendazole,eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox, andnitroimidazole. The skilled artisan is aware of other agents that mayfind utility for the treatment of parasitic disorders.

Embodiments of the present disclosure contemplate the use of a compoundof Formula (I) (e.g., a crystalline form described herein) incombination with agents useful in the treatment or prevention ofbacterial disorders. Antibacterial agents can be classified in variousmanners, including based on mechanism of action, based on chemicalstructure, and based on spectrum of activity. Examples of antibacterialagents include those that target the bacterial cell wall (e.g.,cephalosporins and penicillins) or the cell membrane (e.g., polymyxins),or interfere with essential bacterial enzymes (e.g., sulfonamides,rifamycins, and quinolines). Most antibacterial agents that targetprotein synthesis (e.g., tetracyclines and macrolides) arebacteriostatic, whereas agents such as the aminoglycoside arebactericidal. Another means of categorizing antibacterial agents isbased on their target specificity; “narrow-spectrum” agents targetspecific types of bacteria (e.g., Gram-positive bacteria such asStreptococcus), while “broad-spectrum” agents have activity against abroader range of bacteria. The skilled artisan is aware of types ofantibacterial agents that are appropriate for use in specific bacterialinfections.

Embodiments of the present disclosure contemplate the use of thecompound of Formula (I) (e.g., a crystalline form described herein) incombination with agents useful in the treatment or prevention of fungaldisorders. Antifungal agents include polyenes (e.g., amphotericin,nystatin, and pimaricin); azoles (e.g., fluconazole, itraconazole, andketoconazole); allylamines (e.g., naftifine, and terbinafine) andmorpholines (e.g., amorolfine); and antimetabolies (e.g.,5-fluorocytosine).

Other Therapeutic Modalities. In another embodiment, the presentdisclosure contemplates the use of a compound of Formula (I) (e.g., acrystalline form described herein) in combination with adoptive celltherapy, a new and promising form of personalized immunotherapy in whichimmune cells with anti-tumor activity are administered to cancerpatients. Adoptive cell therapy is being explored usingtumor-infiltrating lymphocytes (TIL) and T cells engineered to express,for example, chimeric antigen receptors (CAR) or T cell receptors (TCR).Adoptive cell therapy generally involves collecting T cells from anindividual, genetically modifying them to target a specific antigen orto enhance their anti-tumor effects, amplifying them to a sufficientnumber, and infusion of the genetically modified T cells into a cancerpatient. T cells can be collected from the patient to whom the expandedcells are later reinfused (e.g., autologous) or can be collected fromdonor patients (e.g., allogeneic).

In certain embodiments, the present disclosure contemplates the use ofthe compound of Formula (I) (e.g., a crystalline form described herein)in combination with RNA interference-based therapies to silence geneexpression. RNAi begins with the cleavage of longer double-stranded RNAsinto small interfering RNAs (siRNAs). One strand of the siRNA isincorporated into a ribonucleoprotein complex known as the RNA-inducedsilencing complex (RISC), which is then used to identify mRNA moleculesthat are at least partially complementary to the incorporated siRNAstrand. RISC can bind to or cleave the mRNA, both of which inhibitstranslation.

The present disclosure encompasses pharmaceutically acceptable salts,acids or derivatives of the agents (and members of the classes ofagents) set forth above.

Dosing

The compound of Formula (I) (e.g., a crystalline form described herein)may be administered to a subject in an amount that is dependent upon,for example, the goal of administration (e.g., the degree of resolutiondesired); the age, weight, sex, and health and physical condition of thesubject to which the formulation is being administered; the route ofadministration; and the nature of the disease, disorder, condition orsymptom thereof. The dosing regimen may also take into consideration theexistence, nature, and extent of any adverse effects associated with theagent(s) being administered. Effective dosage amounts and dosageregimens can readily be determined from, for example, safety anddose-escalation trials, in vivo studies (e.g., animal models), and othermethods known to the skilled artisan.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (themaximum tolerated dose (MTD)) and not less than an amount required toproduce a measurable effect on the subject. Such amounts are determinedby, for example, the pharmacokinetic and pharmacodynamic parametersassociated with ADME, taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the compound of Formula (I) (e.g., acrystalline form described herein) may be an amount that, whenadministered in one or more doses to a subject, produces a desiredresult relative to a healthy subject. For example, for a subjectexperiencing a particular disorder, an effective dose may be one thatimproves a diagnostic parameter, measure, marker and the like of thatdisorder by at least about 5%, at least about 10%, at least about 20%,at least about 25%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, or more than 90%, where 100% is defined as thediagnostic parameter, measure, marker and the like exhibited by a normalsubject.

In certain embodiments, the compound of Formula (I) (e.g., a crystallineform described herein) may be administered (e.g., orally orparenterally) at dosage levels of about 0.01 mg/kg to about 50 mg/kg, orabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic effect.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 1 to 1000milligrams of the active ingredient, particularly 1, 3, 5, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and1000 milligrams of the active ingredient. In some embodiments, thecompositions contain from 25 milligrams to 350 milligrams of the activeagent. In some embodiments, the compositions contain 50 milligrams. Insome embodiments, the compositions contain 100 milligrams of the activeagent. In some embodiments, the compositions contain 300 milligrams ofthe active agent.

For parenteral administration of a compound of formula (I), the compound(e.g., a crystalline form described herein, or a lyophilized formthereof) can be provided prior to its reconstitution in a suitablevehicle. In some embodiments, the compound of formula (I) is provided inan amount of 1 to 1000 milligrams, particularly 1, 3, 5, 10, 15, 20, 25,50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000milligrams of the active ingredient. In some embodiments, the compoundof Formula (I) is provided in an amount of 25 milligrams to 350milligrams. In some embodiments, the compound is provided in an amountof 25 milligrams to 120 milligrams. In some embodiments, the compound isprovided in an amount of 25 milligrams to 110 milligrams. In someembodiments, the compound is provided in an amount of 25 milligrams to100 milligrams.

In some embodiments, the compound of Formula (I) (e.g., a crystallineform described herein) may be administered (e.g., orally orparenterally) on a monthly, weekly or daily basis. In some embodiments,the compound of Formula (I) may be administered at least once a month,such as twice a month, three times a month, four times a month, once aweek, or daily. In some embodiments, the compound of Formula (I) may beadministered once every week, once every two weeks, once every 3 weeks,once every 4 weeks, once every 5 weeks, or once every 6 weeks. Incertain embodiments, the compound of Formula (I) may be administered(e.g., orally) one or more times a day. In some embodiments, thecompound of formula (I) may be administered (e.g., orally) 1, 2, or 3times a day. In some embodiments, the compound of Formula (I) may beadministered (e.g., orally) once a day. In some embodiments the compoundof Formula (I) may be administered (e.g., parenterally) 1, 2, 3, or 4times a month. In some embodiments, the compound of Formula (I) may beadministered (e.g., parenterally) once every other week.

In certain embodiments, the oral formulation comprising a compound ofFormula (I) (e.g., a crystalline form described herein) is administeredsuch that a dose of between 50 mg and 350 mg of the crystalline form ofa compound of Formula (I), such as 50 mg, 75 mg, 100 mg, 125 mg, 150 mg,175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, or 350 mg isadministered daily. In one embodiment, the oral formulation isadministered such that a dose of 100 mg of the compound of Formula (I)is administered daily. In another embodiment, the oral formulation isadministered such that a dose of 300 mg of the compound of Formula (I)is administered daily.

In certain embodiments, the dosage of the compound of Formula (I) (e.g.,a crystalline form described herein) is contained in a “unit dosageform”. The phrase “unit dosage form” refers to physically discreteunits, each unit containing a predetermined amount of the compound ofFormula (I) (e.g., a crystalline form described herein), either alone orin combination with one or more additional agents, sufficient to producethe desired effect. The predetermined amount of the compound of Formula(I) in the unit dosage form can be equal to the desired dosage, or afraction thereof. For example, the unit dosage form can comprise thedesired dose or ½, ⅓, ¼, ⅕, ⅙, 1/7, or ⅛ of the desired dose. In certainsuch embodiments, the unit dosage form can be administered 1, 2, 3, 4,5, 6, 7 or 8 times, respectively, to achieve the desired dose of theactive ingredient. In one or more embodiments, the predetermined amountof the compound of Formula (I) in the unit dosage form is equal to or is½ of the desired dose. In certain such embodiments, the unit dosage formis administered 1 or 2 times, respectively, to achieve the desired doseof the active ingredient. It will be appreciated that the parameters ofa unit dosage form will depend on the particular agent and the effect tobe achieved.

VIII. Kits

The present disclosure also contemplates kits including a compound ofFormula (I) (e.g., a crystalline form described herein), andpharmaceutical compositions thereof. The kits are generally in the formof a physical structure housing various components, as described below,and may be utilized, for example, in practicing the methods describedabove.

A kit can include a compound of Formula (I) disclosed herein (providedin, e.g., a sterile container), which may be in the form of apharmaceutical composition suitable for administration to a subject. Thecrystalline form of the compound of Formula (I) can be provided in aform that is ready for use (e.g., a tablet or capsule) or in a formrequiring, for example, reconstitution or dilution (e.g., a powder)prior to administration. When the crystalline form of the compound ofFormula (I) is in a form that needs to be reconstituted or diluted by auser, the kit may also include diluents (e.g., sterile water), buffers,pharmaceutically acceptable excipients, and the like, packaged with orseparately from the crystalline form of the compound of Formula (I).When combination therapy is contemplated, the kit may contain theseveral agents separately or they may already be combined in the kit.Each component of the kit may be enclosed within an individualcontainer, and all of the various containers may be within a singlepackage. A kit of the present disclosure may be designed for conditionsnecessary to properly maintain the components housed therein (e.g.,refrigeration or freezing).

A kit may contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampule, tube or vial).

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium. In some embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g., via the internet, are provided.

IX. Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present disclosure, and are not intended to limit thescope of what the inventors regard as their disclosure, nor are theyintended to represent that the experiments below were performed or thatthey are all of the experiments that may be performed. It is to beunderstood that exemplary descriptions written in the present tense werenot necessarily performed, but rather that the descriptions can beperformed to generate data and the like of a nature described therein.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.), but some experimental errors anddeviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: min=minute(s); h or hr=hour(s);equiv=equivalents; mg=milligram; g=gram; ml or mL=milliliter; 1 orL=liter; mM=millimolar; M=molar; HPLC=high performance liquidchromatography; NMR=nuclear magnetic resonance; XRPD=x-ray powderdiffraction; DSC=differential scanning calorimetry; DVS=dynamic vaporsorption; RH=relative humidity; HPT=heptane; EtOAc=ethyl acetate;EtOH=ethanol; DCM=dichloromethane; MTBE=methyl tert-butyl ether;MEK=methyl ethyl ketone.

LC: Agilent 1100 series; Mass spectrometer: Agilent G6120BA, single quad

LC-MS method: Agilent Zorbax Eclipse Plus C18, 4.6×100 mm, 3.5 mM, 35°C., 1.5 mL/min flow rate, a 2.5 min gradient of 0% to 100% B with 0.5min wash at 100% B; A=0.1% of formic acid/5% acetonitrile/94.9% water;B=0.1% of formic acid/5% water/94.9% acetonitrile

Flash column: ISCO Rf+

Reverse phase HPLC: ISCO-EZ; Column: Kinetex 5 mm EVO C18 100 A;250×21.2 mm (Phenomenex)

X-Ray Powder Diffraction (XRPD)

XRPD analysis was carried out on a PANalytical X'pert pro, scanning thesamples between 3 and 35° 2θ. The material was gently ground to releaseany agglomerates and loaded onto a multi-well plate with Kapton or Mylarpolymer film to support the sample. The multi-well plate was then placedinto the diffractometer and analyzed using Cu K radiation (α1 λ=1.54060Å; α2=1.54443 Å; β=1.39225 Δ; α1:α2 ratio=0.5) running in transmissionmode (step size 0.0130° 2θ) using 40 kV/40 mA generator settings.

Differential Scanning Calorimetry (DSC)

Approximately 5 mg of material was weighed into an aluminum DSC pan andsealed non-hermetically with a pierced aluminum lid. The sample pan wasthen loaded into a Mettler Toledo DSC-3, heated and held at 30° C. untila stable heat-flow response was obtained. Once a stable heat-flowresponse was obtained, the sample and reference were heated to 450° C.at a scan rate of 5° C./min and the resulting heat flow responsemonitored. Nitrogen was used as the purge gas, at a flow rate of 50cm³/min.

Dynamic Vapor Soprtion (DVS)

Approximately 10 mg of sample was placed into a mesh vapor sorptionbalance pan and loaded into a DVS-1, DVS Intrinsic or DVS Advantagedynamic vapor sorption balance by Surface Measurement Systems. Thesample was subjected to a ramping profile from 40-90% relative humidity(RH) at 10% increments, maintaining the sample at each step until astable weight had been achieved (dm/dt 0.004%, minimum step length 30min, maximum step length 500 min) at 25° C. After completion of thesorption cycle, the sample was dried using the same procedure to 0% RHand then a second sorption cycle back to 90% RH. Two cycles wereperformed. The weight change during the sorption/desorption cycles wereplotted, allowing for the hygroscopic nature of the sample to bedetermined. XRPD analysis was then carried out on any solid retained.

Example 1: Preparation of crystalline Forms A and B of[({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonicacid

Step 1: The heterocycle (25 g, 133 mmol) and ammonium sulfate (175 mg, 1mol %) were charged in a 1 L round bottom flask equipped with a magneticstir bar. HMDS (133 mL, 1M) was added and the mixture was refluxed for 4hours under an air atmosphere (heating block temperature 155° C.).Excess HMDS was evaporated under vacuum at 60° C. and then the flask wasplaced under high vacuum at 45° C. for 30 minutes. This operation wasrepeated to make sure all excess HMDS was removed.

The pale orange oil residue was dissolved in anhydrous MeCN (266 mL) andthe sugar (46.55 g, 146.3 mmol) was added. The resulting mixture wasstirred until all the sugar was dissolved (typically 5 min., yellowsolution). TMSOTf (4.8 mL, 26.6 mmol) was then added dropwise over 20minutes (slight exotherm). Upon completion of the TMSOTf addition, LCMSanalysis showed that all the starting heterocycle was consumed. Thereaction was then stirred for 17-20 hours (deeper colored mixture). AnLCMS aliquot showed greater than 90% UV purity with a ratio between thedesired product and its glycosidic epimer of 97:3. EtOAc (350 mL) andNaHCO_(3(sat.)) (300 mL) were successively added at which point themixture turned deep blue. The layers were separated and the aqueouslayer was extracted once with EtOAc (150 mL). The combined organiclayers were dried over Na₂S₂O₃, filtered and evaporated to dryness. Thedeep blue oil was dissolved in DCM (300 mL). Silica (50 g) and activatedcharcoal (15 g) were added and the resulting suspension was stirredvigorously for 1.5 hours. It was then filtered over Celite to deliver aclear yellow pale to colorless solution. The filtrated was evaporated todryness to deliver the crude material. The clear oil was dissolved inEtOAc (1.33 mL/g). The solution was stirred vigorously and hexanes (4.5mL/g) was added at which point a cloudy mixture is obtained. The mixturewas heated to reflux until complete dissolution, cooled to roomtemperature and seeded with seed crystals. After 1 hour at roomtemperature the mixture was placed in a fridge (0° C.) for 20 hours. Thecrystals were then filtered and rinsed with cold MTBE (2×80 mL+1×50 mL)to yield pure product (46.85 g, 79%). The mother liquors were evaporatedto dryness and the crystallization procedure was repeated. It deliveredadditional material (4.45 g, 7%). Global yield is 51.3 g, 86%.

Step 2: A 3-neck 5 L round-bottomed flask fitted was charged with asolution of the product from step 1 (157 g, 353 mmol) in dimethylsulfoxide (353 mL, 1 M). To the solution was added(1S)-1-(2-fluorophenyl)ethylamine.HCl (93 g, 529 mmol, 1.5 equiv)followed by triethylamine (170 mL, 1.2 mol, 3.5 equiv). The reactionmixture was heated to 80° C. and stirred with an overhead mechanicalstirrer for 48 h. The mixture was cooled to room temperature and dilutedwith methanol (700 mL, 0.5 M). K₂CO₃ (233 g, 1.2 mol, 3.5 equiv) wasadded and the reaction stirred at room temperature. After 40 h, thereaction mixture was filtered through celite and the filter cake waswashed with methanol (2×200 mL). The solution was concentrated in vacuoto remove volatiles. To the remaining solution was added 3.5 L of waterwhile stirring vigorously. The resulting precipitate was then collectedand washed with water (3×1 L) to afford the desired product as a tansolid (139 g, 92%).

Step 3: To a solution of the product from step 2 (45.74 g, 108 mmol) and2,2-dimethoxypropane (66.3 ml, 541 mmol) in acetone (270 mL) at roomtemperature was added p-TsOH (2.05 g, 10.8 mmol). The reaction wasstirred for two hours then concentrated under reduced pressure. Thecrude amber oil was reconstituted in EtOAc (1.0 L) and washed withsaturated NaHCO₃ (500 mL). The organic layer was separated and stirredwith activated charcoal then filtered. The filtrate was dried overNa₂SO₄, filtered and concentrated in vacuo to provide an off-whitesolid. The solid was suspended with 1:1 EtOAc:hexanes (500 mL) andcollected via vacuum filtration. The filter cake was washed with hexane(100 mL) then dried under high vacuum to afford the desired product as awhite solid (39.2 g, 78%).

Step 4: To a suspension of methylenebis(phosphonic dichloride) (81.0 g,324 mmol, 3.0 equiv) in THF (162 mL, 2.0 M) at 0° C. was addedN,N-diisopropylethylamine (20.7 mL, 119 mmol, 1.1 equiv). To theresulting mixture was added a solution of the product from step 3 (50.0g, 108 mmol, 1.0 equiv) in THF (347 mL, 0.31 M) dropwise over the courseof 1 h. Following addition, the resulting mixture was stirred at 0° C.for an additional 15 minutes, then the solution was transferred viacannula to a pre-cooled (0° C.) flask containing 0.2 M HCl (1080 mL).The reaction mixture was warmed to 30° C. and stirred at 30° C. for 16 h[acetonide deprotection]. Upon completion, the reaction mixture waswashed with 1.5:1 MTBE/THF (5×900 mL). The aqueous phase was dilutedwith brine (960 mL) and extracted with 2:1 2-MeTHF/THF (1 L). Theorganic phase was collected and then washed with brine (2×500 mL). Tothe organic layer was added DOWEX Marathon C H⁺ Form (20 g/L, 20 g) andstirred for 2 hours at room temperature. The DOWEX beads were removed byfiltration and the resulting solution was concentrated under reducedpressure to afford a colorless/off-white foam (crude Formula (I)).

To purify the compound via recrystallization, the solid was dissolved inEtOH (313 mL) with stirring and then CH₃CN (1,175 mL) was added over thecourse of 5 minutes. The resulting clear solution was stirred at 25° C.for 1 h, during which time crystallization occurred. The mixture wasallowed to sit at 25° C. for 12 h, and then the white solid wascollected by vacuum filtration, rinsed with 6:1 CH₃CN/EtOH (150 mL), anddried under reduced pressure at 55° C. for 4 days to afford the productas a white solid (32.6 g, 52% yield, 98.5% UV purity, containing 0.75wt. % CH₃CN). The isolated solid was identified as crystalline Form B byXRPD (see WO 2020/123772).

A sample of crystalline Form B was subsequently dried in a vacuum ovenat 60° C. for 16 h, resulting in the isolation of crystalline Form A asdetermined by XRPD (see WO 2020/123772).

Example 2: Preparation of Crystalline Form I of Formula (I)

A flask was charged with Form A of Example 1 (1.00 g) and denatured EtOH(5 mL, 5 parts). The resulting mixture was heated to ˜55-60° C. to forma first clear solution. Toluene (10.0 mL, 10 parts) was added at atemperature of 55-60° C. to form a second clear solution. The secondclear solution was allowed to cool to room temperature over ˜1.5 hours,and stirred at room temperature overnight (about 18 hours) to form awhite suspension. The white solid was collected by vacuum filtration,rinsed with 1:1 EtOH/toluene (2 parts), and dried at 55-60° C. undervacuum for 3 days to afford the Form I as a white solid (0.82 g, 82%yield, containing 0.04 wt % ethanol and about 0 wt % toluene).

The crystalline Form I was characterized by an XRPD pattern as shown inFIG. 1 and was further characterized by a differential scanningcalorimetry (DSC) thermogram as shown in FIG. 2.

Similar results are observed using Form B as the starting material.

Example 3: Alternative Preparation of Crystalline Form I of Formula (I)

A flask was charged with 3.5 g of Form A or Form B and suspended in 17.5mL of anhydrous absolute EtOH. The reaction mixture was heated to 35-40°C., resulting in a clear solution. Next, 35 mL of EtOAc was added to thesolution portion wise over a period of 30 minutes while maintaining thetemperature at 35-40° C. The mixture was subsequently allowed to cool to20-25° C., at which time the solution became a slurry. The slurry wasallowed to stir for 18 hr. The mixture was further chilled to 0-5° C.and maintained at that temperature for 6 hr. The resulting solids werefiltered through a Buchner funnel and suction dried under N₂ for 18 hr.The solids were identified as Form I by XRPD.

Similar results are observed using Form B as the starting material.

Example 4: Preparation of Crystalline Form II of Formula (I)

A flask was charged with Form A of Example 1 (1.00 g) and denatured EtOH(5 mL, 5 parts). The resulting mixture was heated to ˜55-60° C. forabout 15 minutes to form a first clear solution. The first clearsolution was allowed to cool to room temperature over ˜1.5 to 2 hours,and stirred at room temperature for about 72 hours to form a thick whiteslurry suspension. Additional ethanol (2 parts) was added to the thickwhite slurry suspension, and the resulting suspension was reheated to˜55-60° C. to form a second clear solution. The second clear solutionwas allowed to cool to room temperature over ˜1.5 hours, and stirred atroom temperature for about 2 hours to form a heavy white slurrysuspension. The white solid was collected by vacuum filtration, rinsedwith EtOH (2 parts), and dried at 55-60° C. under vacuum for 18 hours toafford the Form II as a white solid (0.37 g, 37% yield, containing 0.68wt % ethanol).

The crystalline Form II was characterized by an XRPD pattern as shown inFIG. 3; and was further characterized by a differential scanningcalorimetry (DSC) thermogram as shown in FIG. 4.

Similar results are observed using Form B as the starting material.

Example 5: Preparation of Crystalline Form III of Formula (I)

A flask was charged with Form A of Example 1 (0.5 g) and denatured EtOH(2.5 mL, 5 parts). The resulting mixture was heated to ˜55-60° C. toform a first clear solution. Methyl tert-butyl ether (5 mL, 10 parts)was added at a temperature of 55-60° C. to form a second clear solution.The second clear solution was allowed to cool to room temperature over˜1.5 hours, and stirred at room temperature overnight (about 18 hours)to form a white suspension. The white solid was collected by vacuumfiltration, rinsed with 2:1 MTBE/EtOH (2 parts), and dried at 55-60° C.under vacuum for 18 hours to afford the Form III as a white solid (0.34g, 68% yield, containing about 0 wt % EtOH and 0.04 wt % methyltert-butyl ether).

The crystalline Form III was characterized by an XRPD pattern as shownin FIG. 5; and was further characterized by a differential scanningcalorimetry (DSC) thermogram as shown in FIG. 6.

Similar results are observed using Form B as the starting material.

Example 6: Preparation of Crystalline Form IV of Formula (I)

A flask was charged with Form A of Example 1 (3.0 g) and anhydrous THF(15 mL, 5 parts). The resulting mixture was heated to ˜55-60° C. to forma first hazy solution. Ethyl acetate (12 mL, 4 parts) was added at atemperature of 55-60° C. to form a second hazy solution. The second hazysolution was allowed to cool to 50-55° C. and seeded with ˜20 mg Form Acrystals of the compound of Formula (I) (as disclosed in WO2020/123772). The resulting mixture was allowed to cool to roomtemperature over ˜1 hour, and seeded again with ˜20 mg Form A crystalsof the compound of formula (I). The resulting mixture was stirred atroom temperature overnight (about 18 hours) to form an off-whitesuspension. The solid was collected by vacuum filtration, rinsed with2:1 THF/EtOAc (6 mL, 2 parts), and dried at 55-60° C. under vacuum for18 hours to afford the Form IV as a white solid (2.8 g, 93% yield,containing 0.05 wt % THF and 0.38 wt % EtOAc).

The crystalline Form IV was characterized by an XRPD pattern as shown inFIG. 7.

Similar results are observed using Form B as the starting material.

Example 7: Preparation of Crystalline Form V of Formula (I)

A flask was charged with Form B of Example 1 (1.00 g) and anhydrous EtOH(5 mL, 5 parts). The resulting slurry was heated to ˜35° C. and allowedto stir for 7 hours to form a clear solution. The mixture was cooled to20-25° C. and stirred for an additional 18 hours, resulting in a whitesuspension. The white solid was collected by vacuum filtration.

The crystalline Form V was characterized by XRPD as shown in FIG. 8, andwas further characterized by a differential scanning calorimetry (DSC)thermogram as shown in FIG. 9. A ¹H NMR of the solid revealed thepresence of 7.6% w/w EtOH, indicating that Form V is an EtOH solvate.

It was subsequently found that Form V slowly converts to Form II whenstored at ambient temperature under an N₂ blanket (>48 hr for fullconversion). Increasing the temperature to 40° C. and drying Form Vunder vacuum increased the conversion rate to Form II (full conversionobserved after 48 hr). Resuspending Form II in a 1:1 EtOH:EtOAc mixtureovernight with stirring allowed the recovery of Form V, demonstratingthat the relationship between Form II and Form V is reversible.

Example 8: Preparation of Form VI

A 20 mL vial was charged with 0.5 g of Form B and 1.5-2.0 mL EtOH. Thesuspension was vigorously stirred at 20-25° C. for 3 days. The solid wascollected by filtration. Crystalline form VI was characterized by XRPD(FIG. 10) and DSC (FIG. 11).

Example 9: Competitive Slurry Experiments Crystalline Form a and Form I

A flask was charged with crystalline Form A (1.5 g) and crystalline FormI (1.5 g) with 30 mL of a 1:2 mixture of ethanol (anhydrous) and ethylacetate. The resulting mixture was allowed to stir at room temperaturefor 5 hours resulting in a thick white slurry. An additional 6 mL of the1:2 EtOH (anhydrous):EtOAc mixture was added. The suspension was allowedto stir for 5 days. A small sample of the suspension was pulled from themixture after 22 hr, 46 hr and 118 hr. The samples were filtered, anddried in an oven at room temperature for 2-3 hr and characterized byXRPD. The results show that Form I is the final form after 46 hr ofstirring, indicating that Form I is more stable than Form A under theseconditions.

Crystalline Form I and Form V at RT

A 20 mL vial was charged with 0.2 g of Form V and 0.2 g of Form I with 4mL of a 1:2 EtOH (anhydrous):EtOAc mixture. The resulting suspension wasstirred vigorously for 3 days. The suspension was filtered, and thesolid was dried under reduced pressure at RT for 3 hr. The isolatedsolids were identified as Form I by XRPD, indicating that Form I is morestable than Form V under these conditions.

Crystalline Form I and Form V at 35° C.

A 15 mL round bottom flask was equipped with a stir bar, thermometer andN₂ inlet, and was charged with 0.25 g Form I and 0.25 g Form V. To theflask was added 6 mL of a 1:2 EtOH (anhydrous):EtOAc mixture was addedto the flask resulting in a white suspension. The suspension was heatedto 35° C. and stirred vigorously for 2 days. The suspension wasfiltered, and the resulting solid dried under reduced pressure at RT for3 hr. The isolated solids were identified as Form I by XRPD, indicatingthat Form I is more stable than Form V under these conditions.

Crystalline Form I and II at RT

A 20 mL vial was charged with 0.2 g Form I and 0.2 g Form II. To thevial was added 4 mL of a 1:2 EtOH (anhydrous):EtOAc mixture, resultingin a uniform suspension. The suspension was stirred vigorously at RT for5 days. The suspension was filtered, and the resulting solid dried underreduced pressure at RT for 3 hr. The isolated solids were identified asForm I by XRPD, indicating that Form I is more stable than Form II underthese conditions.

Crystalline Form I and II at 35° C.

A 15 mL round bottom flask was equipped with a stir bar, thermometer andN₂ inlet, and was charged with 0.2 g Form I and 0.2 g Form II. To theflask was added 5 mL of a 1:2 EtOH (anhydrous):EtOAc mixture was addedto the flask resulting in a white suspension. The suspension was heatedto 35° C. and stirred vigorously for 2 days. The suspension wasfiltered, and the resulting solid dried under reduced pressure at RT for3 hr. The isolated solids were identified as Form I by XRPD, indicatingthat Form I is more stable than Form II under these conditions.

The results of the competitive slurry experiments are summarized inTable 2 below.

TABLE 2 Results of competitive slurry of crystalline forms in a 1:2mixture of EtOH(anhydrous):EtOAc Starting Material Temperature ResultingForm Form A vs Form I RT Form I Form I vs Form V RT Form I Form I vsForm V 35° C. Form I Form I vs Form II RT Form I Form I vs Form II 35°C. Form I

Example 10. Competitive Slurry of Crystalline Form I, II and V at RT

The relative form stability between Forms I, II and V was investigatedby aging mixtures of the mixed forms in EtOH, EtOAc, and mixturesthereof. The Form V starting material contained some Form II asdetermined by its XRPD pattern.

In two 4 mL vials, enough Form I and Form V (containing some Form II)were mixed with 1 mL of the appropriate solvent to generate a slurry ineach vial. The two slurries were combined and shaken at 600 rpm at 20°C. The solids from the slurries were isolated for XRPD characterizationat time 0, 1 day and 2 days (FIG. 12A-D). The results are summarized inTable 3 below.

TABLE 3 Observations from competitive slurry conversion testing at 20°C. End Form by XRPD V % EtOH in Started from Forms I + V + II ExperimentEtOH/EtOAc mixture a 100 Form II b  80 Form II c  67 Form I + Form II(*) d  50 Form I e  33 Form I f  20 Form I + Form V + Form II (*) g  0Form I + Form V + Form II (*) *: after 2 days; slow kinetics.

XRPD patterns of the end solids collected after overnight aging arepresented in FIG. 12A-D. It can be seen that both Forms I and Vconverted to Form II in solvents with ≥80 v % EtOH, while the mixture ofForms I and II remained in 67 v % EtOH system. In 50˜33 v % EtOHsolvents, Form V converted to Form I, while in ≤20 v % EtOH system FormsI and V remained after 2 aging for two days. The lack of conversion inthe ≤20 v % EtOH system was attributed to the low solubility of thecrystalline forms.

Example 11. Solubility Profiles of Forms I, II and V in EtOH/EtOAc

Equilibrium solubilities of Form I and Form V were measured inEtOH/EtOAc at different ratios at 20° C. The Form V starting materialcontains some Form II as determined by XRPD.

In a 4 mL vial, 50 mg of the appropriate solid was mixed with 1 mL ofeach solvent. The resulting slurry was shaken at 600 rpm at 20° C.overnight. The supernatant from each vial was sampled for HPLC forequilibrium solubility, and the solids isolated and characterized byXRPD. The results are plotted in FIG. 13 and summarized in Table 4below.

TABLE 4 Solubility Assessment of Form I and Form II + V in EtOH/EtOAc V% EtOH in Solubility Solubility End Form by # EtOH/EtOAc (mg/mL) (wt %)XRPD Form I Test A1 100 46.54 6.4% Form I B1  80 32.75 4.2% Form I C1 67 23.65 3.0% Form I D1  50 13.71 1.7% Form I E1  33  8.93 1.0% Form IF1  20  1.01 0.1% Form I G1  0  0.00 0.0% Form I Form II + V Test A2 10028.05 3.7% Form II B2  80 33.37 4.2% Form II C2  67 30.88 3.8% Form IID2  50 22.60 2.8% Form II E2  33 13.99 1.7% Forms II + V F2  20  4.680.6% Forms II + V G2  0  0.12 0.0% Forms II + V

The solubility profile shows that ethanol increases solubilities ofForms I and V and EtOAc is an anti-solvent. Maximum solubility of ˜46mg/mL was observed for Form I in pure EtOH. No crystal form change wasobserved in Form I vials. In vials containing a mixture of Forms II andV, Form II was the final form in high ethanol (≥50 v %) systems. Withlow ethanol content (<50 v %), no form change was observed in vialscontaining a mixture of Form II and V.

The data support that a) Form I is more stable than Forms II and V inEtOAc rich solvent system except for too high EtOAc (with too lowsolubility), and b) Form II is more stable than Forms I and V in EtOHrich or EtOAc lean solvent systems.

Example 12: Polymorphic Relationship Map Based on EtOH:EtOAc SolventSystems

A map of the polymorphic relationships between crystalline Form I, IIand V was deduced from observations from EtOH:EtOAc solvent systems(FIG. 14). From the EtOH:EtOAc solvent systems, it has been found thata) EtOAc rich solvent systems favor Form I isolation; and b) EtOAc leanor free solvent systems favor Form II isolation.

Example 13: Solubility Profiles of Forms I, II and V in EtOH:Heptane

Equilibrium solubilities of Forms I, II and V were measured inEtOH/Heptane (HPT) solutions of different ratios at 20 and 35° C.

In 4 mL test vial, the appropriate solids (Form I or Form II containingForm V) were mixed with ˜1 mL of each solvent to generate slurry. Theresulting slurry was shaken at 600 rpm at 20 and 35° C. overnight. Afterovernight aging, supernatant from each vial was sampled for HPLC forequilibrium solubility, solids were isolated to check by XRPD.

FIG. 15 plots the solubility profiles of Forms I and the mixture ofForms II and V in EtOH/HPT at 20 and 35° C. HPT is shown as an effectiveanti-solvent. Form I solubility is higher than the solubility of FormII/V mixtures at all solvent ratios, indicating Form II/V as the moststable form under these conditions. The large solubility differencebetween Forms I and the mixture of Forms II and V under EtOH richcondition implies that isolating Form II/V from EtOH/HPT is easier thanisolating Form I.

Example 14. DVS Characterization of Forms I and II

Form I and II were analyzed by dynamic vapor sorption (DVS) at 25° C.(FIG. 16 and FIG. 17). Form II demonstrated much less water uptake from40% to 70% relative humidity (RH) than Form I. Characterization of thepost-DVS samples by XRPD showed that both forms underwent conversion toForm VI during the experiment.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

1. Crystalline Form I of a compound of Formula (I):

characterized by an X-ray powder diffraction (XRPD) pattern comprisingthree or more peaks at 11.1, 11.6, 13.8, 14.7, 15.4, 16.6, 17.0, 18.6,19.3, 20.1, 21.3, 22.1, 23.0, 24.8, 26.6, 27.3, and 29.1 degrees 2θ(±0.2 degrees 2θ). 2-3. (canceled)
 4. The crystalline Form I of claim 1,characterized by an XRPD pattern comprising peaks at 11.1, 13.8, 18.6,20.1, 23.0, and 24.8 degrees 2θ (±0.2 degrees 2θ), and optionally, oneor more peaks at 11.6, 14.7, 15.4, 16.6, 17.0, 19.3, 21.3, 22.1, 24.8,26.6, 27.3, and 29.1 degrees 2θ (±0.2 degrees 2θ). 5-7. (canceled) 8.The crystalline Form I of claim 1, characterized by an X-ray powderdiffraction pattern that is substantially in accordance with FIG.
 1. 9.(canceled)
 10. The crystalline Form I of claim 1, further characterizedby a differential scanning calorimetry (DSC) thermogram comprising anendothermic peak at about 163.9° C.
 11. The crystalline Form I of claim1, further characterized by a melting point onset of about 155.1° C. asdetermined by a differential scanning calorimetry thermogram (DSC). 12.The crystalline Form I of claim 10, wherein the DSC thermogram issubstantially in accordance with FIG.
 2. 13. Crystalline Form II of acompound of Formula (I):

characterized by an X-ray powder diffraction (XRPD) pattern comprisingthree or more peaks at 10.1, 10.8, 12.8, 13.7, 16.5, 17.7, 19.0, 22.8,and 24.6 degrees 2θ (±0.2 degrees 2θ). 14-15. (canceled)
 16. Thecrystalline Form II of claim 13, characterized by an XRPD patterncomprising peaks at 16.5, 22.8, and 24.6 degrees 2θ (±0.2 degrees 2θ),and optionally, one or more peaks at 10.1, 10.8, 12.8, 13.7, 17.7, and19.0 degrees 2θ (±0.2 degrees 2θ). 17-19. (canceled)
 20. The crystallineForm II of claim 13, characterized by an X-ray powder diffractionpattern that is substantially in accordance with FIG.
 3. 21. (canceled)22. The crystalline Form II of claim 13, further characterized by adifferential scanning calorimetry (DSC) thermogram comprising anendothermic peak at about 166.5° C.
 23. The crystalline Form II of claim13, further characterized by a melting point onset of about 157.4° C. asdetermined by a differential scanning calorimetry thermogram (DSC). 24.The crystalline Form II of claim 22, wherein the DSC thermogram issubstantially in accordance with FIG.
 4. 25-43. (canceled) 44.Crystalline Form V of a compound of Formula (I):

characterized by an X-ray powder diffraction (XRPD) pattern comprisingthree or more peaks at 10.4, 15.1, 15.8, 16.3, 16.8, 18.5, 19.1, 19.7,21.7, 22.1, 23.0, 23.5, 26.0, 26.5, 28.4, 28.9, and 31.4 degrees 2θ(±0.2 degrees 2θ). 45-46. (canceled)
 47. The crystalline Form V of claim44, characterized by an XRPD pattern comprising peaks at 15.8, 16.3,16.8, 18.5, 19.1, 21.7, 22.1, and 23.0 degrees 2θ (±0.2 degrees 2θ), andoptionally, one or more peaks at 10.4, 15.1, 19.7, and 23.6 degrees 2θ(±0.2 degrees 2θ). 48-49. (canceled)
 50. The crystalline Form V of claim44, characterized by an X-ray powder diffraction pattern that issubstantially in accordance with FIG.
 8. 51. (canceled)
 52. Thecrystalline Form V of claim 44, further characterized by a differentialscanning calorimetry (DSC) thermogram comprising an endothermic peak atabout 150.4° C.
 53. The crystalline Form V of claim 44, furthercharacterized by a melting point onset of about 135.8° C. as determinedby a differential scanning calorimetry thermogram (DSC).
 54. Thecrystalline Form V of claim 52, wherein the DSC thermogram issubstantially in accordance with FIG.
 9. 55-64. (canceled)
 65. Apharmaceutical composition comprising a crystalline form of any one ofclaims 1, 13 or 44, and a pharmaceutically acceptable excipient.
 66. Amethod of treating a disease, disorder, or condition, mediated at leastin part by CD73, said method comprising administering an effectiveamount of a crystalline form of the compound of any one of claims 1, 13or 44, to a subject in need thereof.
 67. (canceled)
 68. The method ofclaim 66, wherein the disease, disorder, or condition is cancer. 69.(canceled)
 70. The method of claim 68, wherein the cancer is selectedfrom the group consisting of melanoma, colon cancer, pancreatic cancer,breast cancer, prostate cancer, lung cancer, leukemia, a brain tumor,lymphoma, ovarian cancer, and Kaposi's sarcoma. 71-77. (canceled)
 78. Amethod of treating cancer in a subject, said method comprisingadministering to said subject an effective amount of a crystalline formof the compound of any one of claims 1, 13 or 44, and at least oneadditional therapeutic agent. 79-89. (canceled)